Satoshi Kishino

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection.

We have developed a simple, rapid and highly sensitive method for determining plasma concentrations of ganciclovir and/or acyclovir by using reversed-phase chromatography followed by pulsed amperometric detection. A linear relationship between the amount of ganciclovir (0.05-10 microg/ml plasma) or acyclovir (0.1-20 microg/ml plasma) and peak height ratio was obtained. The relative standard deviations of all standard curves were greater than or equal to 0.999. The limits of detection for ganciclovir and acyclovir quantitation were 10 ng/ml and 50 ng/ml (signal/noise >3), respectively. Daily fluctuations of plasma standard curves (n=5) for the ganciclovir and acyclovir samples were small, with relative standard deviations (RSD) of 3.3 and 4.5% (n=5), respectively. The intra-assay precision for the ganciclovir and acyclovir samples were 6.9 (n=5) and 5.5% (n=5), respectively. Inter-assay precision of ganciclovir (n=3) and acyclovir (n=3) ranged from 2.6 to 6.8% and 3.5 to 5.0%, respectively. Using this method, the pharmacokinetics and removal of ganciclovir during continuous hemodiafiltration (CHDF) in a liver transplant recipient being treated for severe cytomegalovirus infection was investigated. The mean (+/-SD) ratio of ganciclovir concentrations at the inlet and outlet of the dialyzer (C(outlet)/C(inlet)) was 0.56+/-0.09. The areas under the curves of ganciclovir up to 12 h postdosing (AUC(0-->12)) at the inlet and outlet of the dialyzer were 12.54 microg h/ml and 7.16 microg h/ml, respectively. The ultrafiltrate of ganciclovir was 16.6 mg. The terminal elimination half-life (T(1/2)) of ganciclovir during CHDF was 3.6 h. These results demonstrate that CHDF effectively removes ganciclovir. Until formal guidelines have been established, ganciclovir or acyclovir dosage should be adjusted according to the results of monitoring of plasma drug concentration. The method described here is suitable for clinical monitoring of plasma ganciclovir or acyclovir levels in solid organ transplant recipients and for use in studies involving pharmacokinetics.

Effective fluconazole therapy for liver transplant recipients during continuous hemodiafiltration.

Fungal infections are still one of the main causes of death and complications after solid organ and bone marrow transplants. The authors evaluated the effect of continuous hemodiafiltration (CHDF) on the pharmacokinetics of fluconazole in liver transplant recipients. Six liver transplant patients (primary biliary cirrhosis, n = 2; fulminant hepatitis, n = 2; viral hepatitis, n = 2) were enrolled in this study. In one patient not receiving CHDF, the fluconazole levels increased with increasing dosages. In contrast, in patients undergoing CHDF, the dosage of fluconazole was increased from 100 mg/d to 200 mg/d, but fluconazole did not reach the targeted levels. It appears that the targeted trough level cannot be achieved by administration of fluconazole at a dosage of 100 to 200 mg/d during CHDF. A higher dosage (600–1000 mg/d) of fluconazole may be required to achieve the therapeutic drug level in patients undergoing CHDF. In patients undergoing CHDF, fluconazole was given at a dosage of 800 mg/d and reached the targeted levels. In addition, after CHDF, the dosage of fluconazole was decreased to 100 mg/d, and fluconazole reached the near-targeted trough level. These results demonstrate that CHDF removes fluconazole from the blood at an efficiently high rate, resulting in its ineffective blood level. To guarantee safe and effective fluconazole therapy, the trough levels should be monitored routinely during CHDF.

Separation methods for glycoprotein analysis and preparation.

Several chromatographic methods have been developed for the isolation and characterization of glycoproteins. In these methods, affinity chromatography, a single-step method, or combined use with general chromatographic methods have now become essential for the purification of many biologically important glycoproteins, including alpha1-acid glycoprotein, immunoglobulins, ceruloplasmin and erythropoietin. On the other hand, almost all glycoproteins exhibit polymorphism associated with their glycan moieties. This feature is wide-spread and has been observed in natural as well as in recombinant DNA glycoproteins. Recently, several sophisticated techniques--such as electromigration method (high-performance capillary electrophoresis) and chromatographic methods (two-dimensional polyacrylamide gel electrophoresis, high-pH anion-exchange chromatography with pulsed-amperometric detection)--have been introduced for qualitative or quantitative estimation of the microheterogeneity of glycoproteins. For gaining further insight into the structure-function relations for microheterogeneity, preparative chromatographic techniques that can yield sufficient quantities of glycoprotein variants must be developed.

Alpha‐1‐Acid Glycoprotein Concentration and the Protein Binding of Disopyramide in Healthy Subjects

Age‐ and gender‐related changes in serum α1‐acid glycoprotein (AAG) concentration and the serum protein binding of disopyramide were examined after intensive medical examination. Based on the clinical chemistry tests over 51 points, 245 subjects were diagnosed as healthy and 71 subjects (22.5%) revealed an abnormal value for at least one item. In the healthy subjects, serum AAG concentration in men was significantly higher than in women (men, 0.78 ± 0.18 mg/mL, mean ± SD; women, 0.67 ± 0.16 mg/mL). In contrast, there were no significant differences in the AAG concentration between age groups for men and women and in the unbound fraction of disopyramide. Gender changes AAG concentration. Age, however, does not change AAG concentration and the protein binding of the basic drug.

Single-step isolation method for six glycoforms of human α1-acid glycoprotein by hydroxylapatite chromatography and study of their binding capacities for disopyramide

A single-step isolation method for the glycoforms of human serum alpha1-acid glycoprotein (AAG) using a hydroxylapatite column under a gradient elution program was developed. The concentrations of N-acetylneuraminic acid and monosaccharides (fucose, N-acetylglucosamine, galactose and mannose) of six AAG glycoforms were determined by the pulsedamperometric detection method. For each AAG glycoform, significant sex-related differences in carbohydrate content have been observed only for AAG glycoforms two and six, and not for each AAG glycoform. The relationship between the extent of the branch in the glycan chain and the binding capacity to disopyramide were examined. Female AAG contained highly sialylated AAG glycoforms compared to male glycoforms. Conversely, male AAG was rich in the lower sialylated AAG glycoform. Furthermore, it was found that the drug binding capacity decreases with increasing branching of the glycan chain. This suggests that the binding sites of AAG are hindered by a relatively large carbohydrate moiety, such as tetraantennary structures.

Changes in the binding capacity of alpha-1-acid glycoprotein in patients with renal insufficiency.

Summary The elevation of alpha-1-acid glycoprotein (AAG) concentration and the binding characteristics of disopyramide (DP) to AAG in patients with renal insufficiency were investigated. The serum AAG concentration and protein binding of DP in patients were significantly greater than those in healthy subjects. However, in both the serum and the purified AAG, Scatchard analysis showed that the number of binding sites per molecule of AAG in patients was significantly lower than that in healthy subjects, although there was no difference in the dissociation constant (Kd). These results suggest that the AAG induced in renal insufficiency is qualitatively different from normal AAG. Moreover, the change of the unbound DP fraction when DP concentration was increased was larger in the patients than in the healthy controls. Therefore, monitoring of the unbound DP would be important for therapeutic drug monitoring in patients with renal insufficiency.

Purification method for α-1-acid glycoprotein with subsequent high-performance liquid chromatographic determination of monosaccharides in plasma of healthy subjects and patients with renal insufficiency

A simple purification method for human plasma α-1-acid glycoprotein (AAG) using an ion-exchange and hydroxyapatite column was developed. The recovery of the method was found to be high. We also improved a determination method for N-acetylneuraminic acid and monosaccharides in the carbohydrate moiety of AAG by using an ion-exchange column and pulse-amperometric detection. By this method, a composition analysis of the carbohydrate moiety of AAG (N-acetylneuraminic acid, fucose, N-acetyl glucosamine, galactose and mannose) was possible with 1.0 ml of plasma. We compared these carbohydrate concentrations in the AAG of patients with renal insufficiency with those of healthy subjects. In the AAG of the patients, the concentrations of N-acetylglucosamine, galactose and mannose were significantly higher than those in the AAG of the healthy subjects.

Influence of continuous venovenous haemodiafiltration on the pharmacokinetics of tacrolimus in liver transplant recipients with small‐for‐size grafts

Abstract: In adult‐to‐adult living donor liver transplantation (LDLT), the graft volume is inevitably much smaller than the ideal liver mass (standard liver volume) for the recipients metabolic demand. Patients with small‐for‐size grafts are treated with continuous venovenous haemodiafiltration (CVVHD) for the artificial liver support. However, little is known about the influence of CVVHD on the elimination of tacrolimus. The objective of this study was to elucidate the effect of CVVHD on the pharmacokinetics of tacrolimus in recipients of LDLT with small‐for‐size grafts. Three liver transplant recipients (one male and two females) and donors (two males and one female) were enrolled in this study. Blood samples from inflow port and outflow port were obtained on the first day at the start of CVVHD. Whole‐blood concentrations of tacrolimus were measured immediately using the microparticle enzyme immunoassay (MEIA; Abbott Laboratories). There was no significant difference between concentrations of tacrolimus in blood sampled at inflow port and outflow port sites and t1/2‐values of tacrolimus in the three recipients were 29.9, 63.6 and 28.8 h. CVVHD did not cause a decrease in the blood tacrolimus concentration. Adjustment to the dose or dosing interval is not required for patients treated with tacrolimus during CVVHD.

Serum alpha-1-acid glycoprotein and protein binding of disopyramide in patients with congestive heart failure

Congestive heart failure is the end stage of cardiac disease and the haemodynamics in patients become very abnormal. Drug effects may also become modified and some changes have been shown in their pharmacokinetics [1, 2]. The intensity of the pharmacological effects of many drugs is determined by the serum concentration, especially the free concentration. Alpha-l-acid glycoprotein (AAG) binds many basic and neutral drugs. A A G levels were reported to change as a result of physiological and pathophysiological conditions [3-5]. There do not appear to have been reports of the level of A A G in patients with chronic heart failure. Disopyramide binds to A A G and it is used in the treatment of cardiac failure as an antiarrhythmic agent [6]. The serum A A G level and protein binding of disopyramide in vitro at the therapeutic concentrations found in patients with chronic heart failure have been studied. All patients were hospitalized for congenital heart disease, valvular heart disease or congestive cardiomyopathy. All were receiving diuretics and digitalis on admission. A few were also taking captopril. Patients with renal insufficiency (serum creatinine > 1.5 mg. dl1) and those taking drugs reported to bind to A A G were excluded [3, 4]. Sera from 4 men and 4 women of NYHA Class IV were studied on admission and 2 weeks later. A A G concentration was measured using a plate immunoassay (Medical & Biological Lab., Nagoya, Japan). Albumin was measured using an AutoAnalyzer. To examine the free fraction of disopyramide in vitro, each serum sample in which the drug level was adjusted to 5 gg. m1-1, was ultrafiltered through a Centriflow C3 LGC apparatus (Millipore Co., Betford, MA. U. S. A.). The disopyramide concentration was measured by HPLC. In short, 0.1 ml ultrafiltrate was added to 0.1 ml internal standard (nicardipine) and was then injected into the HPLC. The column was a HITACHI 3053 (ODS-18) and it was held at 55 °C. The eluent was 0.2 M acetic ammonium methanol (30 : 70) and the flow rate was 0.7 ml/min. UV detection wave length was 270 nm. Statistical analysis used Students paired t test. The results are summarised in Table 1. The medicines being taken did not change after admission except for increased doses of furosemide. The NYHA class fell from IV to III after treatment. The A A G concentration declined significantly and there was a significant increase in the free fraction of disopyramide (P < 0.05). The albumin concentration did not change. The A A G concentration may change from a normal of 0.5 mg/ml to 3.0 mg/ml in disease [5]. The A A G level here was shown to be increased in patients with severe heart failure. After partial recovery, the A A G concentration fell. Various reasons have been suggested for such a change [5], but the mechanism remains unknown. Sudden death is a significant hazard amongst patients with congestive heart failure. Disopyramide is used for cardiac arrhythmias. Its therapeutic concentration is narrow range 2 ~tg-m1-1 to 5 pg. mlt) and toxic effects occur above 8 gg-m1-1 [6]. Disopyramide is more than 90% bound to A A G and less than 10 % to albumin [6]. Changing the A A G level therefore has significance for this drug and others bound to AAG. The free fraction of disopyramide in vitro showed wide interindividual variation in patients with heart failure. The interindividual variation and A A G concentrationdependent changes in the free drug fraction complicate interpretation of total drug concentration. Therapeutic dose monitoring may be limited when only the total serum

Rapid and simple method for the determination of α1-acid glycoprotein in serum by column liquid chromatography

Abstract A rapid and simple method for the determination of α1-acid glycoprotein (AAG) in serum was developed by using an anion-exchange column for clean-up of serum and a hydroxyapatite column for high-performance liquid chromatography (HPLC). A good correlation was observed between this HPLC method and the conventional radial immunodiffusion method. The method may also be used to determine the AAG concentration in the serum of experimental animals.