Ultrafiltration and subsequent high performance liquid chromatography for in vivo determinations of the protein binding of etoposide.

Etoposide is extensively (approximately 94%) bound to plasma proteins and the free non-protein-bound levels have been shown to correlate more closely to toxicity than total drug concentrations. A rapid and easily performed method, compared to the time consuming equilibrium dialysis, to obtain the free fraction is needed. The aim of this study was to evaluate ultrafiltration and subsequent high performance liquid chromatography (HPLC) for the determination of protein binding of etoposide. Spiked plasma from healthy, drug-free volunteers was used to compare ultrafiltration, using Amicon Centrifree filters, with equilibrium dialysis at 37 degrees C. The variability (CV) of the ultrafiltration method was 6.1 and 13.5% (n = 6) at 37 degrees C and room temperature (RT), respectively. The relative size of the free fraction obtained by ultrafiltration at 37 degrees C and RT was 1.22 (P = 0.0005) and 0.37 (P = 0.0001), respectively, compared with equilibrium dialysis at 37 degrees C. The chromatographic separation of metabolites from the mother compound when free etoposide is analyzed is crucial. It is shown that a hydroxy-acid metabolite of etoposide is quite dominant in a protein-free plasma fraction. The free concentrations were determined throughout a dose interval of 24 h in a patient receiving etoposide 100 mg/m2 daily. Ultrafiltration and subsequent HPLC is considered convenient and suitable for in vivo pharmacokinetic investigations.

Effects of pronounced haemodilution on the plasma protein binding of lidocaine.

The effects of pronounced haemodilution on the protein binding of lidocaine was investigated in vitro in plasma from five healthy adult volunteers. The plasma was diluted with a phosphate buffer to reach a plasma protein concentration normally seen during paediatric cardiopulmonary bypass (CPB) and protein binding was determined at a low (1.5 micrograms/ml) and a moderate (4 micrograms/ml) total plasma concentration of lidocaine. The effects of different haematocrits on plasma protein binding was also determined over the haematocrit range 20-60%. The binding of lidocaine was found to be inversely related to the degree of dilution, i.e. the free fraction increased significantly with increasing dilution (p < 0.0001). Furthermore, the binding was dependent on the total plasma concentration of lidocaine, since a significantly higher percentage of free drug was found at the higher total lidocaine level (4 micrograms/ml) compared with the lower level (1.5 micrograms/ml) (p < 0.05). No significant difference in the free fraction of lidocaine could be found over the studied haematocrit range. The results of the present study indicate that plasma protein levels commonly associated with CPB in neonates and infants are associated with a significant increase in the free, unbound and pharmacologically active fraction of lidocaine compared with normal conditions. The use of commonly recommended dosages of lidocaine might result in toxic-free concentration in this setting.

Protein binding of 2-chloro 2'-deoxyadenosine (cladribine) in healthy subjects and in patients with leukaemia.

The plasma protein binding of 2-chloro-2'-deoxyadenosine (CdA) at 37 degrees C was studied by ultrafiltration in 5 healthy volunteers, in 11 patients with haematological malignancies and in purified protein preparations. In the patients, the binding of CdA to plasma proteins was 25.0% and in healthy subjects it was 21.1%. In a solution of human serum albumin (40 g.l-1), 24.3% CdA was bound, but less than 5% was bound in a solution of alpha 1-acid-glycoprotein (0.7 g.l-1). No dependence of binding on the concentration of CdA was found within a range 25-1000 nmol.l-1. In conclusion, due to its limited binding to plasma proteins, any change in the binding of CdA is unlikely to have a major influence on its pharmacological effect.

Pharmacokinetics of total and free valproic acid during monotherapy in infants.

The pharmacokinetics of free and total valproic acid (VPA) in plasma and whole blood after oral administration during steady state was investigated in seven infants (mean age 10.7 months) receiving monotherapy. The VPA concentrations in whole blood closely followed those in plasma but at a reduced level. A positive correlation was found between dose and mean plasma concentration (r = 0.71). Mean terminal half-lives were similar in plasma and whole blood (12.5 and 15.5 h, respectively), but were considerably longer than for free VPA (6.4 and 6.5 h, respectively; P less than 0.01). There was a significant decrease in half-lives with increasing age (P less than 0.05). Plasma and whole blood clearance for total VPA was higher than reported in older infants and adults (17.8 and 28.9 ml/kg per hour) and was considerably higher for free VPA (127.6 and 188.8 ml/kg per hour, respectively). The increase in clearance compared with that in older subjects is well in concordance with a lower protein binding of VPA (mean 85.3%). Of special importance is that the percentage of unbound VPA increased with increasing concentrations of total VPA. The fraction of unbound VPA in plasma increased even more in subjects with low albumin concentrations (P less than 0.01).

Pharmacokinetics of free and total sodium valproate in adolescents and young adults during maintenance therapy.

The pharmacokinetics of total and free valproic acid (VPA) in plasma and whole blood was investigated in seven adolescents and young adults (mean age 17.3 years) during a dosage interval at steady state. The concentration curves of VPA in whole blood after an oral morning dose (mean 8.2 mg/kg body wt.) closely followed those in plasma but at a reduced level. The apparent volume of distribution (Vd) of total VPA was 0.150-0.197 l/kg body wt. and of free VPA 0.911-1.58 l/kg body wt., which indicates considerable distribution of unbound VPA as well as drug binding to extravascular proteins. The terminal half-life of free VPA (6.4-6.7 h) was significantly shorter (P less than 0.05) than the half-life of total VPA (10.4-11.9 h). The binding of VPA in plasma was concentration dependent and fluctuated considerably within the individual dosage intervals. Concentrations of unbound VPA in plasma water of whole blood varied to a corresponding degree, since distribution to blood cells was low (mean 2.2%). It is concluded that there are substantial differences in the pharmacokinetics of free and total VPA. This may contribute to the well-known poor correlation between dose, plasma concentrations and effect of VPA.

Pharmacokinetics of free and total flucloxacillin in newborn infants.

Flucloxacillin 50 mg/kg b.w. was administered intravenously (in combination with ampicillin/gentamicin) and orally (with amoxicillin) to 9 newborn infants (gestational age 33-41 weeks) to treat bacterial infections. The concentrations of flucloxacillin in plasma and urine after i.v. injection were analysed according to an open two-compartment model, and the plasma protein binding of flucloxacillin and its distribution to blood cells and plasma water in whole blood were determined. Considerable differences were found from values reported in adults. The terminal half-life averaged 4 h 38 min and was significantly correlated with gestational age. Plasma clearance was low (0.744 ml X min-1 X kg-1), due to the small renal clearance (0.182 ml X min-1 X kg-1), whilst non-renal clearance (0.563 ml X min-1 X kg-1) was approximately the same as in adults. The mean apparent volume of distribution of total drug (Vz) was 0.280 l/kg. The corresponding volume of distribution of unbound drug (Vu1 + Vu2) was 1.74 l/kg, which indicates considerable extravascular drug binding. The plasma protein binding of flucloxacillin (mean 86.3%) was significantly correlated with gestational age and the bilirubin/albumin concentration ratio. Bioavailability after oral administration, when corrected for changes in terminal half-life, was 47.7%, which is only slightly lower than that reported in adults. Since the plasma concentrations after both i.v. and oral administration were well above the MIC-values generally reported for Staphylococcus aureus, and since few side-effects were observed, intravenous injection or, in selected cases, orl administration of flucloxacillin appears to be a reliable therapy for the treatment of infections due to sensitive strains of S. aureus in premature newborn infants.

A simplified model for adjustment of gentamicin dosage in newborn infants.

In a retrospective study of 72 neonates during treatment with gentamicin, poor correlation was found between dosage based on body weight and gentamicin serum concentrations. Calculation of adequate gentamicin dosage regimen during steady-state based on individual pharmacokinetic parameters according to Gibaldi & Perrier was then studied in 35 newborn infants during therapy. Predictions were based on gentamicin serum concentrations taken prior to and 1, 3, and 5 hours after the first (n = 8) or second (n = 12) dose (group A), or only prior to and one hour after dose (group B, n = 15). Half-life (t1/2), apparent volume of distribution (Vd), body clearance (Clbody) and elimination rate constant (beta) were not significantly different when calculated after the first or second dose or during steady-state. The correlation between predicted and observed gentamicin concentration was high in both groups (p less than 0.005) and the slopes congruent with unity. After dose or interval correction, 73% of the observed predose concentrations (mean 2.0 micrograms/ml) were within 1 microgram/ml of predicted values. One hour after dose the predicted (mean 5.7 micrograms/ml) and observed (mean 6.2 micrograms/ml) values were not significantly different. Higher precision was noted when the predictions were based on 4 samples (group A) compared to 2 (group B). Since the calculations may be performed by a simple desk calculator rapid advice may be given to the clinical staff on adequate gentamicin dosage even in small severely ill preterm infants.

Pharmacokinetics and distribution of flucloxacillin in pacemaker patients.

The pharmacokinetics of flucloxacillin in plasma and tissue fluid after i.v. infusion of 1 g was analyzed according to an open two-compartment model in 19 patients with bradyarrhythmias (mean age 70.8 years) admitted for implantation or replacement of a permanent pacemaker system. After the first infusion of flucloxacillin (5 min), the distribution phase was rapid (t 1/2 alpha = 0.13 h). The plasma half-life of elimination (t 1/2 beta) was 1.51 h, which is almost twice as long as reported in healthy volunteers. Total plasma clearance (93.1 ml/min) was also lower than is usually found in healthy individuals, due to low renal clearance of flucloxacillin (60.2 ml/min). The total apparent volume of distribution during the beta-phase (Vdarea) was 0.172 l/kg and distribution in the central compartment (Vc) 0.064 1/kg. In each patient plasma protein binding and drug distribution to plasma water, proteins and blood cells in whole blood were determined. Binding in plasma to proteins was 91.0% and distribution to blood cells in whole blood 13.8%. The mean distribution volume of free flucloxacillin during the beta-phase (Vd beta free) was 2.18 1/kg, which exceeds total body water, suggesting possible intracellular distribution and substantial tissue binding. Plasma concentrations of flucloxacillin after the fourth dose (1 g t.i.d.) were very similar to those obtained after the first infusion and those predicted from the single dose kinetics. The concentration of flucloxacillin in fluid from the pacemaker pockets in 5 patients averaged 12.1 micrograms/ml and 9.5 micrograms/ml at 1 and 5 h, respectively, which was more than ten times the MIC-values for Staphylococcus aureus and S. epidermidis.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of phenobarbital in whole blood during pregnancy and perinatally--an in vitro study.

The distribution of phenobarbital (PB) in whole blood and its fractions in 8 pregnant women, in 10 mothers and infant pairs at delivery and in 7 controls was studied by equilibrium dialysis. The lowest protein binding was in cord plasma. A correlation was found between the maternal and cord plasma PB binding. The adult plasma PB binding ratio was correlated with the molar albumin concentration, but no such relation was found in cord plasma, suggesting inhibition of binding by other factors. As a counterpart to the lower protein binding in maternal and cord plasma, the fraction of PB distributed to erythrocytes in those groups was significantly higher. The apparent concentration of PB in maternal erythrocytes was increased. The distribution of drugs to the various fractions of whole blood is an important consideration perinatally, since the binding characteristics of plasma and blood cells in mother and fetus will be crucial determinants for the placental transfer of drugs.

Drug binding to plasma proteins during human pregnancy and in the perinatal period. Studies on cloxacillin and alprenolol.

Plasma protein binding of one acidic drug, cloxacillin, and one basic drug, alprenolol, was determined by equilibrium dialysis at +37 degrees C during pregnancy and the 1st postnatal week in 12 women and their newborn infants and in 7 nonpregnant women (controls). A significant increase in fraction free cloxacillin in maternal plasma occurred during pregnancy already from the 2nd trimester compared to the controls (p less than 0.01) and was most pronounced at delivery (median values 0.126 and 0.069, respectively). A similarly increased fraction free cloxacillin was found in cord blood (median value 0.108) which further increased during the 1st postnatal week (range 0.112-0.164). In maternal plasma the binding capacity returned to the values of the controls during the same time period. The binding of cloxacillin was significantly correlated with the concentration of albumin (p less than 0.01). High correlation was also found between binding of the basic drug alprenolol and concentration of orosomucoid (p less than 0.005). This was most obvious in the newborn infants with low concentrations (range 0.1-0.3 g/l) and in the mothers during the puerperium with high concentrations of orosomucoid (range 0.7-2.5 g/l). On the basis of plasma protein binding data in the mother and her child, a maternal to fetal plasma concentration ratio was calculated. For cloxacillin this ratio was close to unity (1.03), while it was significantly above unity for alprenolol (1.72). At equilibrium, therefore, the total plasma concentration of alprenolol in the mother can be expected to exceed the concentration in her infant.

Drug distribution in whole blood of mothers and their newborn infants: studies of cloxacillin and flucloxacillin.

The distribution of cloxacillin and flucloxacillin in whole blood from seven newborn infants and their mothers was determined in vitro by equilibrium dialysis at 37 degrees C. Seven healthy, non-pregnant women of reproductive age served as controls. The distribution of the penicillins to erythrocytes was the same in the infants as in the adults. It was significantly lower in the presence of plasma albumin than when plasma was replaced by isotonic phosphate buffer. The plasma protein binding of cloxacillin and flucloxacillin in 22 infants was significantly lower than in the controls, but was slightly higher than in the mothers. A significant correlation between binding of cloxacillin and flucloxacillin in the same individual suggested that the two drugs were bound to similar sites. During the first postnatal week binding in infant plasma decreased. This change was correlated with an increase in the bilirubin levels. In the mothers, the binding increased during the first week after delivery. On the basis of the distribution data, maternal to fetal plasma and whole blood concentration ratios at equilibrium were calculated. These ratios were lower for flucloxacillin (medians 0.770 and 0.821, respectively) than for cloxacillin 0.996 and 1.094). Accordingly, at equilibrium somewhat higher levels of flucloxacillin should appear on the fetal than on the maternal side, whereas the concentrations of cloxacillin would be expected to be approximately the same.

Pharmacokinetic aspects of streptomycin treatment of neonatal septicemia.

Patients with suspected neonatal septicemia were treated with ampicillin, cloxacillin, and streptomycin. The plasma concentrations of streptomycin were followed. First, the levels were determined during a full dose interval (12 h) in 11 infants. The results were used for development of a routine system for monitoring the plasma levels in all streptomycin treated newborns. This system, the "3-point check", involved blood sampling at 1, 3, and 5 h after administration during every second dose interval. The results of this routine procedure were evaluated both in a retrospective and prospective study. The "3-point check" gave a sufficient description of the total exposure to streptomycin under routine clinical conditions and continuous information to the physician in charge of the patient about the drug level. In 9 cases of 50, the report from the laboratory resulted in dose change for correction of a too low or too high plasma concentration. The dosage used, 7.5 mg streptomycin intramuscularly every 12th hour, appeared to be satisfactory in most patients. Peak values rarely exceeded 30 microgram/ml and were usually lower than 25 microgram/ml. Almost half of the children had plasma levels below 5 microgram/ml at the end of the dose interval (after 12 h). Although the correlation between pharmacokinetics and clinical outcome is difficult to establish in neonatal sepsis, we suggest that our guiding principle to avoid plasma levels above 25 microliter/ml is reasonable. In 35 out of 78 patients an otological examination of the newborns was performed within 13 months after streptomycin treatment and no signs of hearing defects were noted.

Nasal glioma.

A case of nasal glioma is presented along with a brief review of the artiology, structure and diagnostic possibilities and published in earlier surveys. Considering the extremely benign nature of this type of tumour, we are of the opinion that the surgical measures may be relatively conservative and that mutilating, or grossly disfiguring, procedures should be avoided.