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Biomedical subjects

M Hanano

Publications and source records attributed to M Hanano.

At least 73 records · Page 4Linked to original sources

Facilitated transport of cefodizime into the rat central nervous system.

The disposition of [14C]cefodizime, a nonmetabolizable analog of cefotaxime, in the central nervous system was studied in control and benzylpenicillin-infused rats using in vivo experimental techniques. After i.v. bolus administration of [14C]cefodizime, a concentration gradient was established from the brain extracellular fluid (ECF) to the cerebrospinal fluid (CSF) (i.e., drug penetration into the CSF after i.v. administration can be accounted for by permeation across the cerebral capillaries and diffusion through the brain ECF and across the ependymal surface into the CSF). Kinetic analysis indicated that the cerebrovascular transfer coefficient for plasma unbound [14C]cefodizime in control rats equaled 5.2 x 10(-5) ml/sec/g brain, a value comparable to that for mannitol. In rats which received an i.v. infusion of benzylpenicillin, the cerebrovascular transfer coefficient for [14C]cefodizime was reduced to approximately 1/15th of the control value, resulting in a reduced central nervous system (brain ECF and CSF)/plasma unbound concentration ratio for this drug. After i.c.v. administration, the efflux of [14C]cefodizime from CSF was comparable to that of [3H]mannitol with a small probenecid-sensitive component and was not affected by treatment with benzylpenicillin. These results indicate that the unidirectional influx of [14C]cefodizime into brain ECF and then into the CSF is facilitated by a mechanism shared with other beta-lactam antibiotics.

Animals↗

Consumption coagulopathy associated with left atrial thrombosis.

Three patients with consumption coagulopathy due to left atrial thrombosis associated with mitral valve disease are described. They had hypofibrinogenemia (0.7-1.7 g/L), mild thrombocytopenia (104-117 x 10(9)/L), and elevated fibrinos/fibrin degradation products (FDP) (20-64 micrograms/ml). Two patients had bleeding symptoms, and one of these also had two episodes of transient ischemic attack. One without bleeding symptoms had three episodes of transient ischemic attack and repeated retinal vein thrombosis. In two patients, preoperative anticoagulation with either heparin or nafamostat mesilate was followed by an increase in plasma fibrinogen level from 0.7 to 5.6 g/L and a decrease in FDP from 64 to 8 micrograms/ml in one patient, and fibrinogen from 1.0 to 2.8 g/L and FDP from 40 to 5 micrograms/mL in another patient. The mitral valve replacement and thrombectomy were performed uneventfully, and their coagulopathy disappeared thereafter. These three patients had a lower platelet count and a shorter platelet survival time than another three patients with mitral valve disease of a similar severity but without coagulopathy. Hemostatic evaluation should be performed in patients suspected of intracardiac thrombosis.

Disseminated Intravascular Coagulation↗

Thrombin vs. plasmin generation in disseminated intravascular coagulation associated with various underlying disorders.

In order to assess the thrombin and plasmin generation in vivo in disseminated intravascular coagulation (DIC), plasma levels of thrombin-antithrombin III (ATIII) complex (TAT) and plasmin-alpha 2-antiplasmin (a2AP) complex (PAP) were measured together with standard coagulation and fibrinolytic parameters in 80 patients with DIC. Both TAT and PAP were markedly elevated in patients with DIC. When plotted by the underlying disease categories, differences in the magnitude of the elevations of these complexes were recognized among groups. Patients with acute promyelocytic leukemia (APL) had the highest PAP, the lowest TAT/PAP ratio, low a2AP, and low fibrinogen, indicating that the most excessive fibrinolysis can occur in APL. Similar profiles, although less marked, were observed in patients with other leukemias and vascular diseases. Patients with sepsis showed the highest TAT/PAP ratio and the lowest PAP with no decrease in a2AP or fibrinogen, demonstrating a relatively impaired fibrinolysis. Patients with cancer had a relatively high TAT and high TAT/PAP ratio. In addition, both TAT and PAP were markedly elevated in patients with shock. From these, it was suggested that, although laboratory manifestations in DIC are extremely variable from patient to patient, underlying disorders are, at least in part, responsible for the observed variations. Recognition of this variable activation of coagulation and fibrinolysis would be helpful for the proper management of patients with DIC.

Adolescent↗

Kinetic analysis of the dose-dependent hepatic handling of 1-anilino-8-naphthalene sulfonate in rats.

The dose dependency in the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS), was investigated by measuring the plasma disappearance and biliary excretion in rats. Bulk of the administered ANS distributed into the liver at 10 min after iv bolus injection. The plasma disappearance curves of ANS were then kinetically analyzed based on a two-compartment model, in which the ligand is eliminated only from the peripheral compartment (liver compartment). The total body clearance (CLtot) decreased with increasing dose of ANS. That is, the values of CLtot were 4.06 and 1.98 ml/min/per kg at the doses of 3 and 100 mumol/kg, respectively. The clearances of the uptake and sequestration processes (CLup and CLseq, respectively) for a total ligand were constant irrespective of dose, while the efflux clearance (CLeff) for a total ligand was increased by twofold with increasing dose. A mechanism for the increase in the CLeff value might be explained by a saturation of the ANS binding to the intracellular proteins. The hepatocellular distribution and the binding of ANS to cytosolic proteins were then determined. ANS mainly distributed to the cytosol fraction, and the unbound fraction in the cytosol increased from approximately 0.04 to 0.09 when the cytosolic concentrations of ANS increased from 40 to 900 microM, respectively. In spite of such increase in the unbound fraction in the cytosol, the CLseq values remained unchanged with increasing dose, suggesting that the saturation of sequestration clearance for unbound ANS might occur. Furthermore, the plasma disappearance curves of ANS at various doses were simultaneously analyzed based on three nonlinear kinetic models: Model I is a model incorporating both saturable intracellular binding and saturable sequestration; Model II is a model incorporating only saturable intracellular binding; Model III is the model incorporating only saturable sequestration. Goodness-of-fit evaluated by AIC value was best for Model I. Taken together, the nonlinearity in the plasma clearance of ANS was confirmed to be attributed to saturation of both its binding to cytosolic proteins and sequestration process.

Anilino Naphthalenesulfonates↗

Measurement of cerebral glucose utilization from brain uptake of [14C]2-deoxyglucose and [3H]3-O-methylglucose in the mouse.

Glucose utilization (GU) in the mouse brain in vivo was measured by the simultaneous use of [14C]2-deoxyglucose (2DG), the glucose analogue that can be phosphorylated in the brain, and [3H]3-O-methylglucose (3MG), the nonmetabolizable glucose analogue. Originally, this method was developed by Gjedde et al. (1985) in the rat and in humans. The present study examined the validity of this method in the mouse brain. The effects of urethane and pentobarbital (PB) on GU were also studied. Whereas the distribution volume of 3MG reached a constant value under each condition after 10 min of the tracer circulation, the apparent volume of distribution of 2DG, which increased with time in the awake mice, did not increase so greatly in the anesthetized and hypothermic mice, indicating that the net rate of 2DG phosphorylation is lowered under these conditions. These data were fitted for the conventional three-compartmental model and the values of rate constants of influx (K1), efflux (k2), phosphorylation, and dephosphorylation for 2DG, and K1 and k2 for 3MG were computed by nonlinear least square regression method. No significant difference in the value of K1/k2, the distribution volume of the precursor pool, was observed between 2DG and 3MG, indicating that 3MG can be used to estimate the distribution volume of unmetabolized 2DG in the brain. The values of GU calculated from the values of estimated parameters were not significantly different from those calculated from blood and tissue radioactivities obtained during 10 min after the injection of the tracers in both awake and PB-anesthetized mice. These findings indicate that the double tracer technique is useful for measuring GU in a short duration experiment.

3-O-Methylglucose↗

Effect of various organic anions on the plasma disappearance of 1-anilino-8-naphthalene sulfonate.

The effects of various organic anions on the hepatic transport of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS) were investigated by measuring the plasma disappearance-time profiles in rats. Ten min after the i.v. administration of ANS (3 mumol/kg), various organic anions (60 mumol/kg) were injected in a bolus. Sulfobromophthalein (BSP), bromophenol blue (BPB) and rose bengal (RB) induced a transient increase in the plasma concentration of ANS (the so-called 'counter-transport' phenomena). The effect of rose bengal was somewhat different. After the administration of rose bengal, the plasma concentration of ANS decreased rapidly followed by a gradual increase. On the other hand, after the administration of bilirubin and taurocholate, the transient increases in plasma ANS concentrations were minimal. No effect was observed after the administration of phenolsulfophthalein (PSP) or oleate. The effects of these organic anions on the binding of ANS to rat liver cytosols were examined by equilibrium dialysis. Sulfobromophthalein, bromophenol blue and rose bengal, which yielded an in vivo 'counter-transport' phenomena, markedly inhibited ANS binding to cytosolic proteins. On the other hand, the other organic anions examined had very small, if any, inhibitory effect. The ANS binders in the cytosol were then identified by gel filtration. ANS bound mainly to X and Y (ligandin) fractions in the cytosol. Sulfobromophthalein, which is one of the organic anions exhibiting the in vivo 'counter-transport' phenomenon, remarkably inhibited ANS binding to ligandin fraction. It was thus suggested that the in vivo 'counter-transport' phenomena may be also explained by the enhancement of back diffusion due to the displacement of intracellular binding. In conclusion, one should be more cautious in interpreting data obtained from so-called in vivo 'counter-transport' experiments.

Anilino Naphthalenesulfonates↗

Kinetic analysis of the downregulation of epidermal growth factor receptors in rats in vivo.

We previously clarified the specific binding sites for epidermal growth factor (EGF) in several organs in rats based on in vivo kinetic analysis (D. C. Kim, Y. Sugiyama, H. Sato, T. Fuwa, T. Iga, and M. Hanano. J. Pharm. Sci. 77: 200-207, 1988). In the present study, we have determined the extent of the receptor downregulation and the recovery rate of the available receptors for EGF in several organs in vivo. At the specified times (30 min-24 h) after intravenous administration of excess unlabeled EGF (300 micrograms/kg), the early-phase (less than 3 min) uptake clearances (k1) of the tracer amount of 125I-EGF, which are proportional to the cell-surface available receptor densities, were determined in the liver, kidney, duodenum, jejunum, ileum, stomach, and spleen. As the result, the k1 value in each organ at 30 min after intravenous administration of unlabeled EGF was lowered close to the receptor-independent clearance value, indicating that the cell-surface receptors were almost completely downregulated, and thereafter, the k1 value showed gradual recovery to the control level. Furthermore, the recovery half-lives showed interorgan differences, namely the half-life (20 min) in the liver was much shorter than those (2-4.5 h) in other organs. These results were considered to reflect the processes of the recycling of internalized EGF receptors to the cell-surface or recruitment of new receptors. It was concluded that the recovery rate of the downregulated receptors in the liver, which is most responsible for the plasma clearance of EGF, is much faster than those in other organs.

Animals↗

Internalization of EGF in perfused rat liver is independent of the degree of receptor occupancy.

The internalization rate of epidermal growth factor (EGF) by the perfused rat liver was evaluated to determine whether the internalization rate constant depends on the degree of receptor occupancy. A tracer concentration of 125I-labeled EGF (30 pM) alone or 125I-EGF plus unlabeled EGF (20 nM) was infused into the liver in the single-pass perfusion system at 37 degrees C. At various times (2-20 min), the perfusion medium was switched to medium of pH 3.0, and the radioactivity of 125I-EGF emerging into the outflow (surface-bound EGF) and remaining in the liver (internalized EGF) was determined. At the tracer condition, less than 0.1% of the surface receptor was occupied by 125I-EGF for 20 min perfusion. When excess unlabeled EGF (20 nM) was present in the perfusate and the bulk of the cell surface receptors was occupied, the density of the cell surface EGF receptor after 20 min dropped to 14% of the initial value. The internalization rate constant, defined as the probability of an occupied receptor being internalized per minute, was calculated from the slope of a plot of the amount of internalized EGF vs. the integrated amount of surface receptor-bound EGF with time. The internalization rate constants calculated from the plots with and without unlabeled EGF in the perfusate were 0.21 and 0.33 min-1, respectively, and did not differ significantly (P greater than 0.1). These results indicated that the internalization rate constant of the EGF receptor complex is independent of the degree of receptor occupancy in the liver.

Animals↗

Effect of pregnancy on the disposition of valproate in rats.

The disposition of sodium valproate in pregnant rats was studied comparing with nonpregnant (control) rats. In the pregnant rats, the total plasma clearance decreased significantly (p less than 0.05) from 7.06 ml/min/kg of the control to 5.34 ml/min/kg, whereas the plasma elimination half-life of valproate did not change. The serum unbound fraction (fs) of pregnant rats increased remarkably. The fs of the fetal plasma was lower than that of the maternal serum in spite of the lower albumin (main binding protein for valproate) concentration in the fetal plasma. A non-linear serum (plasma) protein binding was observed both in the control and the fetal rats, but not observed in the pregnant rats. In the pregnant rats, the tissue-to-plasma concentration ratio (Kp) of the brain was higher than that in the control rats, whereas the Kp values of the liver and and lung were lower than those in the control rats. In other tissues, the Kp values did not show a significant difference. Rapid placental transfer was observed and the Kp value of the fetus was 0.43.

Animals↗

Prediction of the pharmacokinetics of cefodizime and cefotetan in humans from pharmacokinetic parameters in animals.

Pharmacokinetic behavior of beta-lactam antibiotics (cefodizime and cefotetan) in humans were predicted from the animal data. Total body clearance (CLp) of these drugs in humans (weighting 65-69 kg) were successfully extrapolated from the allometric relationship between the clearance for the unbound drug in plasma and body weight (r = 0.954-1.000) with a power of 0.948-0.991 for cefodizime and 0.700-0.756 for cefotetan. We also predicted the volume of distribution at steady state (Vdss), the volume of distribution in the central compartment (V1) and the volume of distribution at beta-phase (Vd beta) of these drugs in humans from the observed human plasma unbound fraction, inasmuch as the plasma unbound fraction correlated well (r = 0.913-0.995) with the values of Vdss, V1, and Vd beta among various animal species. Based on these predicted pharmacokinetic parameters, we calculated the plasma concentration profiles of these drugs in humans and found a good agreement between the predicted and observed values. We also report here that the prediction is successful when we consider the plasma protein binding of these drugs.

Animals↗

Decrease in the number of receptors for epidermal growth factor in the liver of D-galactosamine-intoxicated rats.

Hepatic transport of epidermal growth factor (EGF) was studied in D-galactosamine-intoxicated rats by the multiple-indicator dilution (MID) method. The extraction ratio of 125I-labeled EGF in the intoxicated rats, obtained from a model-independent analysis of the dilution curves, decreased to 45% of the control values. A distributed two-compartment model was fitted to the dilution data by nonlinear least-squares regression, and the kinetic parameters, kon.PT (product of on-rate constant and receptor density), koff (off-rate constant) and ks (sequestration rate constant) were determined. The values of kon.PT and ks in the intoxicated rats decreased to approximately one-half and one-third of those in the control rats respectively. Similar decreases in the kon.PT and ks values in the intoxicated rats were also observed for the transport of 125I-labeled insulin, a positive control, into the liver. The 125I-labeled EGF binding experiment at equilibrium using liver homogenates revealed that the intoxication reduced the receptor density (PT) to one-third of the control values, whereas the equilibrium dissociation constant (kd) did not change significantly. The activities of Na+,K+-ATPase, cytochrome P-450 and glutathione S-transferase decreased in the intoxicated rats to 70-80% of the control values. The number of nuclei per unit area of tissue slices was also reduced to 70% of the control. Thus, the extent to which the enzyme activities and the number of nuclei decreased in the intoxicated liver was smaller than that of the number of EGF receptors. It is concluded that the reduction of EGF receptors cannot be explained by the "intact hepatocyte hypothesis" but rather by the functional change of hepatocytes induced by the administration of D-galactosamine.

Animals↗

Effect of procainamide on renal tubular transport of cimetidine in the isolated perfused rat kidney.

The effect of procainamide on renal tubular transport of cimetidine was studied in isolated perfused rat kidney based on the multiple indicator dilution (MID) technique. T-1824-labeled albumin (a vascular reference), [14C]creatine (an extracellular reference), and [3H]cimetidine were rapidly injected into the renal artery of isolated perfused rat kidney in the presence or absence of procainamide (100 microM) in the perfusate, and normalized outflow-time patterns were secured from rapidly sampled renal perfusate. A distributed two-compartmental model was fitted to the dilution data by non-linear least-squares regression, and the influx, efflux and sequestration rate constants were estimated. Net transport and influx processes of cimetidine were competitively inhibited by procainamide (PA), while the efflux and sequestration processes were increased. The increase in the values of the efflux and sequestration rate constants by addition of procainamide may be explained by the increase in the tissue binding of cimetidine. However, these three processes were not significantly affected by p-aminohippurate (PAH). These results suggest that both cimetidine and procainamide are secreted into the lumen by an organic base transport mechanism in the perfused kidney, in which the spatial organization and cell polarity of the kidney are maintained.

Albumins↗

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Anilino Naphthalenesulfonates↗

Kinetic analysis of the elimination process of human epidermal growth factor (hEGF) in rats.

Pharmacokinetic study of human epidermal growth factor (hEGF) in rats was performed in vivo. The hepatic extraction ratio (EH) of [125I]hEGF, determined from the difference between the artery and the hepatic vein plasma concentrations at steady state, was 0.19. The hepatic clearance (CLH:7.56 ml/min/kg body wt), calculated by multiplying EH by the hepatic plasma flow rate (QP,H), was approximately 70% of the total body clearance (CLtot: 10.8 ml/min/kg body wt), which was determined from the steady-state arterial plasma concentration and the infusion rate. These results indicated that the liver is the main organ responsible for the removal of [125I]hEGF from the systemic circulation in rats. The renal extraction ratio (ER) of [125I]hEGF was half of that of [14C]inulin; this may have resulted from the plasma protein binding of [125I]hEGF, which was approximately 50% as determined by the charcoal adsorption method and the equilibrium gel-filtration method. The renal clearance (CLR:2.65 ml/min/kg body wt), calculated by multiplying ER by the renal plasma flow rate (QPR), was approximately 17% of the CLtot (15.6 ml/min/kg body wt), indicating a minor contribution of CLR to CLtot compared with that of CLH to CLtot. The CLR of [125I]hEGF calculated from the urinary excretion data was one-tenth of that calculated from the plasma concentration difference between the femoral artery and the renal vein at steady state. These results suggest that the bulk of [125I]hEGF cleared from the plasma by the kidney may have been metabolized further in the renal tubules before appearing in the urine.

Animals↗

Coagulation studies in thrombotic thrombocytopenic purpura, with special reference to von Willebrand factor and protein S.

The profile of blood coagulation and fibrinolysis was studied in detail in eight patients with acute thrombotic thrombocytopenic purpura (TTP). In the majority of the patients, fibrinogen, factor XIII, antithrombin III, alpha 2-plasmin inhibitor, plasminogen, and alpha 2-macroglobulin were normal, whereas FDP, plasmin-alpha 2-plasmin inhibitor complex, and tissue-type plasminogen activator antigen were marginally or moderately elevated. Low fibronectin values were observed in four patients. Protein C and C4b-binding protein were nearly normal, whereas total protein S and free protein S were reduced in five and six patients, respectively. A positive correlation was found between total protein S and C4 and between free protein S and C3. von Willebrand factor antigen (vWf:Ag) and ristocetin cofactor (RCof) were either normal or elevated, but RCof/vWf:Ag ratio was decreased in seven patients. Crossed immunoelectrophoresis and sodium dodecyl sulfate (SDS)-agarose gel electrophoresis revealed that the large vWf multimers were either absent from or relatively decreased in all patients except one. In addition, one patient had unusually large vWf multimers, and a low-molecular-weight vWf fragment was apparently observed in three patients. These findings indicate that the intravascular generation of thrombin and plasmin was minimal in TTP and suggest that the alterations of the vWf molecule were caused not only by consumption through its participation in platelet thrombus formation but also by accelerated proteolysis. Low protein S values would be related to the immunological abnormalities underlying TTP.

Adolescent↗

Epidermal growth factor as a regulatory hormone maintaining a low pH microclimate in the rat small intestine.

This study was designed to determine the effect of epidermal growth factor (EGF) in the lumen on the pH of the intestinal surface in the rat jejunum, which is referred to as "microclimate-pH". In the control experiment, a significant pH gradient was observed between the mucosal surface (approximately pH 6.8) and the bulk phase (approximately pH 7.3). The microclimate-pH was decreased by 0.2-0.6 pH units after addition of higher concentrations of EGF (3-100 nM) to the lumen. The microclimate-pH thus decreased recovers to the control value by replacing EGF with TES buffer, suggesting that the EGF effect is reversible. Considering that the Na+-H+ exchanger exists on the luminal membrane of the intestinal cells, the decrease in the microclimate-pH which was induced by EGF added to the luminal side may be due to the activation of Na+-H+ exchanger.

Animals↗

Relationship between in vivo first-order intestinal absorption rate constant and the membrane permeability clearance.

An attempt was made to explore the quantitative relationship between the intestinal absorption data obtained from in vivo studies and in situ perfusion studies. The time course of the fraction remaining to be absorbed of L-glucose, erythritol, and urea in the small intestine following the intrajejunal administration to rats was described by a one-compartment model. Thus derived first-order intestinal absorption rate constants (ka) obtained from the in vivo studies in rats were compared with the membrane permeability clearances (CLa,m) estimated in a single-pass perfusion system. Not only were ka and CLa,m in the same increasing order of L-glucose less than erythritol less than urea, but also the operational luminal volumes given as CLa,m/ka were in agreement with the in vivo luminal volume of jejunum estimated by an inulin dilution method. This result suggests that the in vivo intestinal absorption rate (or ka) can be correlated with the intestinal membrane permeability (or CLa,m) by taking the in vivo luminal volume into account.

Animals↗

Receptor-mediated transport of peptide hormones and its importance in the overall hormone disposition in the body.

A remarkable feature of the pharmacokinetics of polypeptide hormones is the contribution of specific binding sites (receptors) to the polypeptide hormone distribution and clearance in the body. The concept of "transport receptor" is now well established, and receptor-mediated endocytosis (RME) is recognized as a general mechanism in the uptake of biologically important peptide hormones. This article focuses on the kinetic analysis of the RME of polypeptides, based mainly upon the observations of the kinetics of epidermal growth factor in the liver. The following points are emphasized: (1) How can we determine the existence and the kinetic constants of polypeptide RME in vivo and in the perfused liver system? A liver perfusion method, the single-pass multiple-indicator dilution technique, has been shown to be suitable for analyzing the dynamics of interaction of peptide hormones with their cell surface receptors. (2) What is the importance of down-regulation of transport receptors to the overall kinetics of polypeptides in vivo? Time profiles of polypeptide plasma concentrations and their surface receptors in the liver after iv administration of epidermal growth factor were simulated with a physiologic pharmacokinetic model that includes kinetic constants representing the interaction of polypeptides and their receptors.

Animals↗