Hitoshi Sato

Inhibitory effects of quinolone antibacterial agents on gamma-aminobutyric acid binding to receptor sites in rat brain membranes.

The specific binding of 3H-labeled gamma-aminobutyric acid ([3H]GABA) to synaptic plasma membranes from rat brains was inhibited by various quinolonecarboxylic acid derivatives (quinolones), and these inhibitions were concentration dependent. The binding of [3H]muscimol to GABAA sites was also inhibited. These inhibitory potencies differed widely among the quinolones examined. The Dixon plots showed that a newly developed difluorinated quinolone, NY-198 [1-ethyl-6,8-difluoro-1,4-dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3- quinolinecarboxylic acid hydrochloride], competitively inhibits the receptor bindings of [3H]GABA and [3H]muscimol. In conclusion, our findings suggest that the inhibition of GABA binding to receptors (including uptake sites) in the brain may be involved in the induction of epileptogenic neurotoxicities by quinolones.

Determination of erythromycin, clarithromycin, roxithromycin, and azithromycin in plasma by high-performance liquid chromatography with amperometric detection

In this study, a high-performance liquid chromatographic method was developed for the quantitative determination of erythromycin (EM), roxithromycin (RXM), and azithromycin (AZM) in rat plasma with amperometric detection under a standardized common condition using clarithromycin (CAM) as an internal standard. This method was also proved to be applicable for the determination of CAM by employing RXM as an internal standard. Each drug was extracted from 150 microl of plasma sample spiked with internal standard under an alkaline condition with tert.-butyl methyl ether. The detector cell potential for the oxidation of the drugs was set at +950 mV. The linearity of the calibration curves were preserved over the concentration ranges of 0.1-10 microg/ml for EM and RXM, and 0.03-3.0 microg/ml for CAM and AZM. Coefficients of variation and relative error were less than 9% and +/-7%, respectively. The analytical method presented here was proved to be useful for the investigation of the pharmacokinetic characteristics of EM, CAM, RXM, and AZM in rats.

Comparative Pharmacodynamic Analysis of Q-T Interval Prolongation Induced by the Macrolides Clarithromycin, Roxithromycin, and Azithromycin in Rats

ABSTRACT In order to evaluate the arrhythmogenic potency of macrolide antibiotics in a quantitative manner, we analyzed the influence of clarithromycin (CAM), roxithromycin (RXM), and azithromycin (AZM) on Q-T intervals from pharmacokinetic and pharmacodynamic points of view and in comparison with the potency of erythromycin (EM) previously reported by us for rats. Male Sprague-Dawley rats were anesthetized, and CAM (6.6, 21.6, and 43.2 mg/kg of body weight/h), RXM (20 and 40 mg/kg/h), and AZM (40 and 100 mg/kg/h) were intravenously injected for 90 min to obtain the time courses of drug concentrations in plasma and the changes in the Q-T intervals during and after the drug injections. Distinct Q-T interval prolongation of up to 10 ms was observed with CAM at its clinical concentrations. RXM and AZM evoked Q-T interval prolongation at concentrations higher than their clinical ranges. The potencies for Q-T interval prolongation, assessed as the slope of the concentration-response relationship, were 6.09, 0.536, and 0.989 ms · ml/μg for CAM, RXM, and AZM, respectively. There was hysteresis between the change in the Q-T intervals and the time course of the plasma concentration of each drug. The rank order of clinical arrhythmogenicity was estimated to be EM > CAM > RXM > AZM, as assessed from the present results and our previous report for EM. In conclusion, RXM and AZM were estimated to be less potent at provoking arrhythmia than EM and CAM. These results should be useful for making a safer choice of an appropriate agent for patients with electrocardiographic risk factors.

Physiologically based pharmacokinetic modeling as a tool for drug development

Since the pioneering work of Haggard and Teorell in the first half of the 20th century, and of Bischoff and Dedrick in the late 1960s, physiologically based pharmacokinetic (PBPK) modeling has gone through cycles of general acceptance, and of healthy skepticism. Recently, however, the trend in the pharmaceuticals industry has been away from PBPK models. This is understandable when one considers the time and effort necessary to develop, test, and implement a typical PBPK model, and the fact that in the present-day environment for drug development, efficacy and safety must be demonstrated and drugs brought to market more rapidly. Although there are many modeling tools available to the pharmacokineticist today, many of which are preferable to PBPK modeling in most circumstances, there are several situations in which PBPK modeling provides distinct benefits that outweigh the drawbacks of increased time and effort for implementation. In this Commentary, we draw on our experience with this modeling technique in an industry setting to provide guidelines on when PBPK modeling techniques could be applied in an industrial setting to satisfy the needs of regulatory customers. We hope these guidelines will assist researchers in deciding when to apply PBPK modeling techniques. It is our contention that PBPK modeling should be viewed as one of many modeling tools for drug development.

Electrochemical evaluation of chemical selectivity of glutamate receptor ion channel proteins with a multi-channel sensor

A new method for evaluating chemical selectivity of agonists towards receptor ion channel proteins is proposed by using glutamate receptor (GluR) ion channel proteins and their agonists N-methyl-D-aspartic acid (NMDA), L-glutamate, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (L-CCG-IV). Integrated multi-channel currents, corresponding to the sum of total amount of ions passed through the multiple open channels, were used as a measure of agonists selectivity to recognize ion channel proteins and induce channel currents. GluRs isolated from rat synaptic plasma membranes were incorporated into planar bilayer lipid membranes (BLMs) formed by the folding method. The empirical factors that affect the selectivity were demonstrated: (i) the number of GluRs incorporated into BLMs varied from one membrane to another; (ii) each BLM contained different subtypes of GluRs (NMDA and/or non-NMDA subtypes); and (iii) the magnitude of multi-channel responses induced by L-glutamate at negative applied potentials was larger than at positive potentials, while those by NMDA and L-CCG-IV were linearly related to applied potentials. The chemical selectivity among NMDA, L-glutamate and L-CCG-IV for NMDA subtype of GluRs was determined with each single BLM in which only NMDA subtype of GluRs was designed to be active by inhibiting the non-NMDA subtypes using a specific antagonist DNQX. The order of selectivity among the relevant agonists for the NMDA receptor subtype was found to be L-CCG-IV > L-glutamate > NMDA, which is consistent with the order of binding affinity of these agonists towards the same NMDA subtypes. The potential use of this approach for evaluating chemical selectivity towards non-NMDA receptor subtypes of GluRs and other receptor ion channel proteins is discussed.

Sustained QT prolongation induced by tacrolimus in guinea pigs.

Recently, clinical cases have been reported of QT prolongation and torsades de pointes associated with the use of tacrolimus (FK506). We examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs in order to evaluate the arrhythmogenicity of FK506 in comparison with quinidine (QND). FK506 (0.1 or 0.01 mg/hr/kg) or QND (30 mg/hr/kg) was intravenously infused to guinea pigs and time profiles of drug concentration in blood and QTc interval were examined during and after infusion. Both FK506 and QND evoked a significant QTc prolongation, and the dose-response relationship showed an anti-clockwise hysteresis, FK506-induced QTc prolongation persisted throughout the duration of the experiment despite a decline in the plasma FK506 concentration, whilst QND-induced QTc prolongation disappeared as plasma concentrations decreased. FK506 induced a sustained QTc prolongation in guinea pigs at drug concentrations in blood that correspond to its therapeutic range in human, suggesting that it might be of clinical significance to monitor the electrocardiogram, especially when patients have congenital or acquired QT-prolonging risk factors.

Binding of radioiodinated human β-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).

Receptor-mediated endocytosis of A14-125I-insulin by the nonfiltering perfused rat kidney

The mechanism of insulin uptake and/or degradation in the peritubular circulation of the kidney was investigated using nonfiltering perfused rat kidneys, in which glomerular filtration was sufficiently reduced. After perfusion of A14-125I-insulin in the nonfiltering kidney for designated intervals, the acid-wash technique was employed to separately measure the acid-extractable and acid-resistant A14-125I-insulin, which were quantitated by HPLC and TCA-precipitability. HPLC profiles showed that the nonfiltering kidney metabolizes A14-125I-insulin only to a small extent during 1-h perfusion, suggesting that the peritubular clearance of A14-125I-insulin was not due to extracellular degradation but for the most part to uptake by the kidney. Acid-extractable A14-125I-insulin rapidly increased with time and reached pseudo-equilibrium with perfusate at approx. 10 min, whereas acid-resistant A14-125I-insulin increased continuously. An endocytosis inhibitor, phenylarsine oxide, inhibited significantly the acid-resistant A14-125I-insulin with no change in acid-extractable A14-125I-insulin, suggesting that the peritubular uptake of A14-125I-insulin largely represents endocytosis of the peptide into the intracellular space. Moreover, both the acid-extractable and acid-resistant A14-125I-insulin were significantly decreased in the presence of unlabeled insulin (1 microM). These lines of evidence suggest that insulin is taken up by the nonfiltering perfused kidney via receptor-mediated endocytosis (RME), which possibly occurs at the basolateral side of renal tubular cells, and that the peritubular clearance of insulin is largely accounted for by this mechanism.

Secretion of interleukin-6 and vascular endothelial growth factor by spindle cell sarcoma complicating Castleman's disease (so-called 'vascular neoplasia')

So‐called ‘vascular neoplasia’ (VN) is a rare tumour of unknown origin that complicates hyaline vascular type Castlemans disease (CD). This paper reports a case of VN complicating CD of hyaline vascular type, in which neoplastic cells were shown to secrete interleukin‐6 (IL‐6) and vascular endothelial growth factor (VEGF). In this case, VN first occurred in the retroperitoneum of a 60‐year‐old male. The lesion showed typical morphology, with three distinct areas: (1) a lymph node‐like area with regressively transformed lymph follicles showing hyaline vascular changes and with a hypervascular interfollicular region filled with slit‐like vascular channels; (2) an area composed of spindle cell sarcoma; and (3) an area showing angiolipomatous hamartoma. A proportion of the cells in the spindle cell area showed severe pleomorphism. Subcutaneous recurrence after 8u2009months was composed purely of pleomorphic spindle cells. A karyotypic analysis of the recurrent tumour showed 47, XXY with some instability. Supernatant from primary culture contained high levels of IL‐6 and VEGF, suggesting high secretion of these cytokines from neoplastic cells. Immunohistochemically, p53 overexpression was identified only in the pleomorphic spindle cells of the primary lesion and metastatic tumour. No features suggestive of vascular origin were shown on immunohistochemical or electron microscopic analysis of the neoplastic cells. Human herpesvirus type 8 was not detected by immunohistochemistry or PCR analysis. High levels of IL‐6 and/or VEGF have been reported to play a role in CD. This is the first case report that clarifies the site of such cytokine production, showing the possibility of CD as a paraneoplastic phenomenon. Copyright

Application of HPLC in disposition study of A14-125I-labeled insulin in mice.

To describe quantitatively the in vivo distribution and elimination of insulin, high-performance liquid chromatography (HPLC) separation was applied to the pharmacokinetic study of human insulin labeled with 125I at tyrosine A14 (A14-125I-insulin) as a tracer. Intact A14-125I-insulin levels were determined by HPLC and trichloroacetic acid (TCA) precipitation in plasma and various tissues after its intravenous bolus injection into mice. TCA precipitation consistently overestimated the intactness of A14-125I-insulin compared with HPLC, possibly due to the presence of both a TCA-precipitable intermediate degradation product of labeled insulin found in HPLC elution profiles and reported high-molecular-weight forms of labeled insulin in plasma. Thus, TCA precipitation gave a considerably lower total plasma clearance (Cltot) value than HPLC. The half-life of A14-125I-insulin was prolonged by a simultaneous injection of 8 U/kg unlabeled insulin, and labeled insulin behaved similarly to [14C]inulin (an extracellular fluid marker). The concentration time profiles of HPLC-separated labeled insulin in plasma were analyzed by a noncompartmental moment method, and both Cltot and steady-state apparent volume distribution (VDSS) of A14-125I-insulin were considerably decreased by unlabeled insulin coadministration. In particular, VDSS of labeled insulin decreased by 79%, similar to that of inulin (181 ml/kg), suggesting that the nonspecific binding of labeled insulin to tissues was so small that VD88 of labeled insulin was reduced to the extracellular fluid volume (∼20% of the body weight) when its receptor binding was blocked effectively by unlabeled insulin. This observation, together with the 63% reduction of CI,tot by unlabeled insulin coadministration, demonstrated that saturable, receptor-mediated processes of distribution and elimination are essentially involved in the pharmacokinetics of HPLC-separated A14-125I-insulin.