Empagliflozin, a novel selective sodium glucose cotransporter-2 (SGLT-2) inhibitor: characterisation and comparison with other SGLT-2 inhibitors.

AIMS: Empagliflozin is a selective sodium glucose cotransporter-2 (SGLT-2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT-2 inhibitors. METHODS: [(14)C]-alpha-methyl glucopyranoside (AMG) uptake experiments were performed with stable cell lines over-expressing human (h) SGLT-1, 2 and 4. Two new cell lines over-expressing hSGLT-5 and hSGLT-6 were established and [(14)C]-mannose and [(14)C]-myo-inositol uptake assays developed. Binding kinetics were analysed using a radioligand binding assay with [(3)H]-labelled empagliflozin and HEK293-hSGLT-2 cell membranes. Acute in vivo assessment of pharmacokinetics was performed with normoglycaemic beagle dogs and Zucker diabetic fatty (ZDF) rats. RESULTS: Empagliflozin has an IC(50) of 3.1 nM for hSGLT-2. Its binding to SGLT-2 is competitive with glucose (half-life approximately 1 h). Compared with other SGLT-2 inhibitors, empagliflozin has a high degree of selectivity over SGLT-1, 4, 5 and 6. Species differences in SGLT-1 selectivity were identified. Empagliflozin pharmacokinetics in ZDF rats were characterised by moderate total plasma clearance (CL) and bioavailability (BA), while in beagle dogs CL was low and BA was high. CONCLUSIONS: Empagliflozin is a potent and competitive SGLT-2 inhibitor with an excellent selectivity profile and the highest selectivity window of the tested SGLT-2 inhibitors over hSGLT-1. Empagliflozin represents an innovative therapeutic approach to treat diabetes.

Do unusual site-specific population dynamics of rodent reservoirs provide clues to the natural history of hantaviruses?

Between January 1995 and November 1997, longitudinal mark-recapture studies of rodent hosts of hantaviruses in a disturbed microhabitat within a shortgrass prairie ecosystem in southeastern Colorado (USA) were conducted. The site was distinguished by edaphic and floristic characteristics unique to this area and associated with historical land use patterns, as well as the year-around availability of water from a functioning windmill. Populations of two common rodent species that are hosts for hantaviruses, Peromyscus maniculatus and Reithrodontomys megalotis, had unusually rapid turnover, a younger age structure, and a much lower prevalence of antibody to Sin Nombre virus than did populations at nearby sites in more typical shortgrass prairie and canyon habitats. Based on these findings, we suggest that a stable resident population of the reservoir is critical to the maintenance of hantaviruses at a given site, and we hypothesize that long-lived, persistently infected rodents are the principal transseasonal reservoir of hantaviruses.

Metabolism and distribution of [2,3-(14)C]acrolein in lactating goats.

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.

Metabolism and distribution of [2,3-(14)C]acrolein in laying hens.

The metabolism and distribution of [2,3-(14)C]-acrolein were studied in 10 laying hens orally administered 1.09 mg/kg of body weight/day for 5 days. Eggs, excreta, and expired air were collected. The hens were killed 12-14 h after the last dose and edible tissues collected. The nature of radioactive residues was determined in tissues and eggs. All of the identified metabolites were the result of the incorporation of acrolein-derived radioactivity into normal natural products of intermediary metabolism in the hen except for 1,3-propanediol, which is a known degradation product of glycerol in bacteria.

Metabolism and distribution of [2,3-14C]acrolein in Sprague-Dawley rats. II. Identification of urinary and fecal metabolites.

The metabolites of [2,3-14C]acrolein in the urine and feces of Sprague-Dawley rats were identified after either intravenous administration in saline at 2.5 mg/kg or oral administration by gavage as an aqueous solution as either single or multiple doses at 2.5 mg/kg or as a single dose of 15 mg/kg. Selected urine and feces samples were pooled by sex and collection interval and profiled by combinations of reverse-phase, anion-exchange, cation-exchange, and ion-exclusion high-performance liquid chromatography (HPLC). Feces were also profiled by size-exclusion chromatography. Metabolites were identified by comparison with well-characterized standards by HPLC and by mass spectrometry. The urinary metabolites were identified as oxalic acid, malonic acid, N-acetyl-S-2-carboxy-2-hydroxyethylcysteine, N-acetyl-S-3-hydroxypropylcysteine, N-acetyl-S-2-carboxyethylcysteine, and 3-hydroxypropionic acid. The fecal radioactivity from the oral dose groups was partitioned into methanol-soluble, water-soluble, and insoluble radioactivity, some of which could be liberated by dilute acid hydrolysis. HPLC analysis of these extracts revealed no discrete metabolites. Size-exclusion chromatography indicated a molecular weight range of 2,000 to 20,000 Da for the radioactivity, which was unaffected by hydrolysis at reflux with 6 M acid or base. This radio-activity was thought to be a homopolymer of acrolein, which was apparently formed in the gastrointestinal tract. The pathways of acrolein metabolism were epoxidation followed by conjugation with glutathione, Michael addition of water followed by oxidative degradation, and glutathione addition to the double bond either following or preceding oxidation or reduction of the aldehyde. The glutathione adducts were further metabolized to the mercapturic acids.

Metabolism and distribution of [2,3-14C]acrolein in Sprague-Dawley rats.

The metabolism and disposition of [2,3-14C]acrolein was studied in Sprague-Dawley rats after oral or intravenous dosing. Four groups of ten rats (five male and five female) were dosed with radiolabeled acrolein intravenously at 2.5 mg kg-1 (Group 2), orally by gavage at 2.5 mg kg-1, either as a single dose (Group 3) or after 14 daily doses of unlabeled acrolein (Group 4), or orally by gavage at 15 mg kg-1 (Group 5). Urine, feces, expired air and organic volatiles were collected for 7 days, after which the animals were sacrificed and tissues collected. All samples were analyzed for total radioactivity. After 7 days, the excretory patterns of male and female rats were almost identical. Urinary excretion was highest in the intravenously dosed animals (66-69%) and lowest in the Group 5 animals (36-40%), whereas the reverse was true for feces (< 2% for i.v. Group 2 animals and 28-30% for the Group 5 animals). Carbon dioxide expiration was comparable (26-31%) across all groups. Tissue concentrations of radioactivity were minimal in all groups (< 1.2%), but concentrations of radioactivity were highest in the intravenous Group 2 animals. The time course of excretion for all groups was similar with the exception of the high-dose animal group, which showed a pronounced delay in excretion during the first 12 h.

Abnormal membrane physical properties of red cells in McLeod syndrome.

McLeod red cells (RBCs) lack Kx antigens and have weak expression of the Kell antigens. Individuals who carry the McLeod phenotype have acanthocytic RBCs and a compensated hemolytic state. To elucidate the role of the protein on which the Kx antigens reside in maintaining membrane deformability, the rheologic properties of McLeod RBCs were determined by ektacytometry. RBCs were obtained from normal individuals and from four patients with McLeod syndrome. Osmotic gradient deformability profiles of McLeod RBCs showed decreased whole cell deformability. Resealed ghosts from McLeod RBCs also showed decreased deformability, partly because of the decreased cell surface area and partly because of an intrinsic membrane stiffness in this syndrome. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytomer, and deformability was recorded continuously as the deformable ghosts fragmented into rigid spherical vesicles. Membranes from McLeod RBCs showed a noticeable increase in mechanical stability. Acquired causes of acanthocytosis, such as liver disease, did not cause the rheologic abnormalities observed in McLeod cells. Other abnormalities noted in McLeod RBCs were decreased RBC potassium content and an increased number of dense RBCs, as determined by centrifugation on a discontinuous density gradient. The data indicate that McLeod RBCs are rigid and have decreased surface area and that their membranes are intrinsically rigid with increased mechanical stability. These abnormalities may account for the reduced RBC survival observed in McLeod syndrome. The protein that carries the Kx surface antigen seems to be required for the maintenance of the normal physical function of RBC skeletal proteins.

Lymphocytic alveolitis in primary HIV infection.

Primary infection with the human immunodeficiency virus (HIV-1) has been associated with a self-limited illness resembling acute infectious mononucleosis. Pulmonary manifestations have been notably absent in published reports. The authors describe a 28-year-old homosexual male who presented with primary HIV-1 infection associated with CD8+ lymphocytic alveolitis. Diagnosis was delayed because HIV antibody was not detected by the Abbott ELISA, although the same and subsequent specimens were later found to be positive by Genetic Systems' ELISA and Western blot analysis. Lymphocytic alveolitis must be added to the expanding clinical spectrum of acute HIV-1 infection. The time to detection of seroconversion may vary with different immunoassays.

Explaining the risks of blood transfusion to patients.

Many patients express concern about the risk of an infection from blood transfusion. Blood transfusion is one of the safest therapies available, but its risks should never be trivialized when talking with patients. The most common infectious complication is hepatitis C, which occurs in 2% to 4% of transfused patients. Hepatitis B occurs in fewer than 1% of such patients. The risk of HIV infection from a blood transfusion is less than 1 in 100,000 in the United States. Explanation of risks is most effective when comparisons are meaningful and phrased from the patient's point of view.

Computer-assisted audits of blood component transfusion.

Comprehensive review of clinical blood transfusion practice at a tertiary-care medical center is complicated by the extraordinary number of patients that receive such therapy. Computer-assisted review of the key objective data used in making the decisions about transfusion is necessary to evaluate the process. Use of 15,873 units of red blood cells, 3,641 units of plasma, 2,619 pools of platelets or pheresis units, and 259 pools of cryoprecipitate was screened by comparing pre-transfusion and post-transfusion blood counts with the medical staff's evaluation criteria. On this basis, 81.4% of transfusion episodes (TEs) were considered fully justified. Medical records were selected for audit from the cases in which the transfusion decisions could not be justified by on-line information. Abstracted data subsequently justified 82 of 139 audited cases; 68.4% of the comments pertaining to the remaining 57 cases adequately explained the transfusion decision. Thus, nearly 96% of the TEs were justifiable as determined by peer review.

Disposition of 8-methoxypsoralen in the rat. Induction of metabolism in vivo and in vitro and identification of urinary metabolites by thermospray mass spectrometry.

The pharmacokinetics and metabolism of 8-methoxypsoralen (8-MOP) were measured in the catheterized rat after pretreatment for 3 days with phenobarbital (PB), beta-naphthoflavone (BNF), 8-MOP, or vehicle. After an iv injection of 10 mg/kg of [14C]8-MOP, timed blood samples were collected and analyzed using a sensitive and specific assay for [14C]8-MOP. Total body clearance of 8-MOP increased from 0.55 +/- 0.06 liter/kg/hr in control rats to 5.6 +/- 0.4, 2.7 +/- 0.4, and 1.2 +/- 0.0 liters/kg/hr in rats pretreated with BNF, PB, and 8-MOP, respectively, indicating that all three compounds are inducers of 8-MOP metabolism. The pattern of urinary metabolites was altered by the enzyme inducers. The urinary excretion of the sulfate conjugate of 5-hydroxy-8-methoxypsoralen was increased from 10 to 40% of the dose after pretreatment with PB. This intact conjugate was identified using thermospray and fast atom bombardment mass spectrometry. Pretreatment with 8-MOP and BNF increased 2- and 3-fold, respectively, the urinary excretion of a labile sulfate conjugate of 5,8-dihydroxypsoralen. Metabolism of 8-MOP was demonstrated in the 9000 g supernatant and microsomes of rat liver and shown to be inducible by pretreatment of rats with BNF, PB, and 8-MOP. 8-MOP was metabolized in incubations with liver microsomes at rates of 0.22 +/- 0.06, 0.38 +/- 0.06, 0.78 +/- 0.07, and 0.91 +/- 0.03 nmol/min/mg of protein for vehicle, 8-MOP-, PB-, and BNF-pretreated rats, respectively. Results of our investigation indicate that the success of therapy with 8-MOP may be influenced by pharmacokinetic interactions with other drugs.

Phenytoin removal during plasma exchange.

Plasma exchange is currently being used to treat a variety of disorders including immune complex and hematologic disorders. It has been shown that the removal of plasma removes drugs bound to plasma proteins. This case documents the removal of phenytoin during plasma exchange therapy. Total and free phenytoin serum concentrations were obtained before and after each exchange. Aliquots were obtained from each pass, and total phenytoin concentrations were determined. The total phenytoin serum concentration increased during the first exchange, while the total concentration decreased as a result of the second exchange. It was determined that approximately 27.7 mg and 30.4 mg of phenytoin were removed by the first and second plasma exchanges, respectively.

Disposition of 8-methoxypsoralen in the rat: methodology for measurement, dose-dependent pharmacokinetics, tissue distribution and identification of metabolites.

The pharmacokinetics and metabolism of 8-methoxypsoralen (8-MOP) were measured in the catheterized rat after a single i.v. dose. Blood samples were collected serially and analyzed using a sensitive and specific assay for [14C]-8-MOP. Total body clearance of 8-MOP was 7.3, 3.9, 1.7, 1.0, 0.78 and 0.42 liters/kg/hr at doses of 0.2, 1.0, 2.5, 5.0, 10 and 20 mg/kg, respectively. The decline in total body clearance indicates that elimination of 8-MOP is dose-dependent in the rat. After i.v. administration of 10 mg/kg of 8-MOP, 71 and 26% of the dose was recovered within 72 hr in the urine and feces, respectively. Unchanged 8-MOP accounted for less than 1% of the excreted radioactivity. In tissue distribution studies at 0.5, 2 and 5 hr after i.v. administration, 8-MOP distributed rapidly to all tissues and concentrated in the fat and kidneys. The concentration of 8-MOP in the skin was 0.4 to 0.6 times that in the blood. Eleven metabolites of 8-MOP were detected in the urine. The metabolites identified after enzymatic hydrolysis were 8-hydroxypsoralen; 5-hydroxy-8-methoxypsoralen; 5,8-dihydroxypsoralen; 5,8-dioxopsoralen; 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid and 8-MOP (formed by ring closure of a coumaric acid metabolite). Thus, these studies indicate that 8-MOP is metabolized in the rat by 1) O-demethylation; 2) hydroxylation at position 5; 3) hydrolysis of the lactone ring and 4) oxidation of the furan ring, a pathway already confirmed in insects, dogs and humans.

Comparison of indium 111-labeled platelets v leukocytes in a pyogenic abscess.

This study sought to determine which blood component, WBCs or platelets, is the more specific indicator of an abscess and where each localizes. An abscess was created using stool in the hind limb of dogs. After 24 hours, one group was given autologous indium 111-labeled platelets and another group was given autologous indium 111-labeled WBCs. Blood, abscess fluid, infected operative control muscle tissue, and nonoperative control muscle tissue were counted for radioactivity 24 hours after administration of the labeled cells. There was significantly (P less than .001) less WBC radioactivity in blood and more within abscess fluid compared with platelets. The highest platelet activity occurred in muscle tissue adjacent to the abscess (P less than .002) compared with platelet activity in abscess fluid or control muscle tissue. The unwanted high platelet blood background activity and the desirable high concentration of WBC radioactivity within the abscess fluid makes the latter the preferential radionuclide imaging agent.

Improved method for the determination of aspirin and its metabolites in biological fluids by high-performance liquid chromatography: applications to human and animal studies.

An improved method has been developed for the determination of acetylsalicylic acid, salicylic acid, gentisic acid, and salicyluric acid in plasma and urine of rabbits and man. Samples are extracted with dichloromethane containing mephenytoin as an internal standard, the solvent is evaporated under reduced pressure, the residue reconstituted and analyzed by high-performance liquid chromatography. Extraction efficiencies, linearity and assay precision were determined. This method has been applied to human bioavailability studies and the data are presented.

Salicylate removal during plasma exchange in normal volunteers.

The removal of salicylate by plasma exchange in normal subjects was studied. Six healthy men volunteers were given aspirin 975 mg every six hours for five days before undergoing plasma exchange. Blood samples for determination of serum salicylate concentrations were obtained from each subject before the 0730 aspirin dose on study days 3-6 and at hourly intervals for several hours before and after plasma exchange, which was performed at approximately 1500 on study day 5. Blood cell counts, serum albumin concentrations, and other blood chemistry values were also evaluated. Salicylate concentrations in serum and in plasma removed by exchange were determined spectrophotometrically. In each subject, serum salicylate concentrations before and after plasma exchange were compared, as were total area under the serum concentration-time curve and salicylate clearance on and off plasma exchange. The mean trough salicylate concentration at 1930 after plasma exchange on day 5 was significantly lower than the mean trough concentration at 1330 before plasma exchange on the same day. The mean AUC during the six-hour period in which plasma exchange occurred was significantly smaller than the AUC in the six-hour period before plasma exchange. Serum albumin concentrations decreased by a mean of 1.7 g/dl as a result of plasma exchange; however, the mean +/- S.D. amount of salicylate removed was only 191.0 +/- 52.4 mg. The amount of salicylate removed by plasma exchange in this study does not appear to be clinically important.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of transfusion-associated cytomegalovirus infection in very low-birthweight infants using frozen blood and donors seronegative for cytomegalovirus.

During a 15-month period, 34 low birthweight infants (less than 1300 g) lacking maternal antibody to cytomegalovirus (CMV) received transfusions from an average of 10 donors per infant. Blood products consisted of predominantly washed deglycerolized frozen red cells from donors lacking antibody to CMV (an average of nine seronegative donors per infant). None of these infants acquired CMV infection while hospitalized. The absence of acquired CMV infections in these infants was in marked contrast (p less than 0.001) to the incidence of CMV infections in seronegative low-birthweight infants during the previous 21-month period when 28 percent acquired CMV (7 of 25 infants). The infants in this previous group received transfusions from an average of 11 donors per infant with an average of four seropositive donors per infant. These results confirm that hospital-acquired CMV infections can be prevented for very low-birthweight infants by donor selection and/or blood processing.

Gamma scintigraphic localization of platelets labeled with indium 111 in a focus of infection.

To determine if autologous platelets would localize in a focus of infection, a pyogenic abscess was created in the left hind limb of dogs, using previously processed human stool, while an identical surgical procedure without bacterial inoculation was performed on the right hind limb. Autologous platelets labeled with indium 111 (500 microCi) were administered intravenously to five control dogs that had not undergone surgery, to eight dogs two hours following stool inoculation, and to five dogs 24 hours after stool inoculation. A statistically significant scintigraphic increase in tracer activity was apparent within 24 hours in each animal at the site of abscess creation. Tissue samples, obtained at 48 hours after the administration of labeled platelets, revealed a significant increase in percent dose of 111In per gram of infected muscle compared with control muscle. These studies show that platelets localize at the site of bacterial infection.