Roger L. Nation

A simple method for the assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of colistin in human plasma. Derivatization with 9-fluorenylmethyl chloroformate was performed in the same solid-phase extraction C18 cartridge used for sample pre-treatment, followed by reversed-phase HPLC with fluorimetric detection. Quantification was achieved using the ratio of the summed peak areas of colistin A and B derivatives to that of the derivative of netilmicin (internal standard). Linear calibration curves were obtained within the concentrations of colistin sulfate from 0.10 to 4.0 mg/l in plasma. Accuracy was within 10% and reproducibility (RSD) was less than 10%.

Bioequivalence requirements for generic products

Many countries have established procedures for the introduction of generic pharmaceutical products. In order to protect consumers, these generic products must be demonstrated to be therapeutically equivalent to a previously approved product, typically an innovator product. The therapeutic equivalence of a generic and an innovator product is most commonly based on the demonstration of bioequivalence, i.e. clinically insignificant differences in the rate and extent of drug absorption usually assessed from pharmacokinetic measurements. This article reviews the bioequivalence requirements for generic products and, in the interest of promoting international harmonisation, highlights those areas where differences exist among countries.

Pharmacokinetics of L‐carnitine in patients with end‐stage renal disease undergoing long‐term hemodialysis

L‐Carnitine is an endogenous molecule involved in fatty acid metabolism. Secondary carnitine deficiency may develop in patients with end‐stage renal disease undergoing long‐term hemodialysis because of dialytic loss. In these patients L‐carnitine can be administered to restore plasma and tissue levels. The objective of this study was to evaluate the pharmacokinetics of intravenous L‐carnitine in patients undergoing long‐term hemodialysis.

Chirality in New Drug Development Clinical Pharmacokinetic Considerations

ConclusionsThere are several clinical pharmacokinetic considerations that are pertinent to the decision to develop either an individual enantiomer or the racemate. In the majority of cases, these considerations favour proceeding down the enantiomer pathway. In the future, it is likely that very few new chiral drugs will be developed as racemates and those that are will come under rigorous scrutiny by the regulatory authorities. As commented recently by Cayen,[41] a member of the pharmaceutical industry, ‘Pharmaceutical companies are very much aware that, in the 1990s, racemate development will be cost ineffective and time-consuming. Regulatory agencies are asking the right questions and taking a very hard look at racemate NCEs (New Chemical Entities); the justification must be convincing. Therefore, why ask for trouble?’.

Effects of cyclophosphamide and adriamycin on rat hepatic microsomal glucuronidation and lipid peroxidation

The effect of cytotoxic drug administration, as a single dose i.p. to rats (six rats/treatment group), on hepatic microsomal UDP-glucuronosyltransferase (UGT) activity was investigated. Glucuronidation of morphine in microsomes from control rats apparently involved at least two enzymes. Administration of cyclophosphamide (CP; 200 mg/kg 7 days prior to killing) significantly increased the rate of morphine glucuronidation over the range 0.05-10 mM, and significantly increased the apparent Vmax for the high capacity isoenzyme from 1.25 +/- 0.12 to 1.95 +/- 0.39 nmol/mg/min. In contrast, the activity of 1-naphthol UGT was not significantly altered by administration of CP. Rats treated with the same dose of CP 1 day prior to killing showed a significant decrease in microsomal morphine-UGT activity at 0.05 and 2.5 mM morphine, but a significant increase in activity was observed following administration of CP or Adriamycin (AD; 10 mg/kg) 4 days prior to killing. The extent of microsomal lipid peroxidation was significantly increased in microsomes obtained from rats treated with CP or AD 4 days prior to killing, and was positively correlated (P less than 0.001) with the rate of glucuronidation of 0.05 and 2.5 mM morphine. Preincubation of microsomes in the presence of CP (5 mM) and AD (100 microM) significantly decreased the rate of glucuronidation of 2.5 mM morphine. In vitro NADPH-mediated lipid peroxidation significantly increased the activity of both the high and low affinity morphine-UGT isoenzymes. Administration of the cytotoxic drugs CP and AD may alter microsomal morphine-UGT activity via the process of lipid peroxidation, although other mechanisms cannot be excluded.

Meperidine binding in maternal and fetal plasma

Meperidine protein binding was measured in nine pairs of maternal and fetal plasma samples obtained at delivery. For the maternal samples, percent bound and binding ratio (bound/free, B/F) were 63.3 ± 6.18% (SD) and 1.79 ± 0.45, and for the fetal samples the corresponding values were 51.7 ± 4.53% and 1.09 ± 0.21. In each case the binding was higher in the mother than in the fetus (p < 0.01). Plasma α1‐acid glycoprotein (α1‐AGP) concentrations were higher (p < 0.01) in maternal than in fetal samples, and there was a correlation between meperidine B/F and plasma α1‐AGP concentration for the maternal and fetal samples (r = 0.752, p < 0.01). Binding studies with purified α1‐AGP showed that this was a cause‐effect relationship. The transplacental binding differential was attributable partially to the maternal‐fetal difference of plasma α1‐AGP concentrations. Meperidine was 17.5 ± 0.35% bound in a 3.5 gm/100 ml solution of human serum albumin; however, there was an inverse correlation (r = −0.798, p < 0.01) between B/F and plasma albumin concentration for the maternal and fetal samples. A relatively large proportion (75%) of the overall variability in B/F was accounted for by plasma α1‐AGP and albumin. Plasma binding of this basic drug was not greatly influenced by the perinatal levels of bilirubin and nonesterified fatty acids. The common clinical observation of greater fetal than maternal plasma total meperidine concentrations at delivery is not the result of more extensive protein binding in fetal than in maternal plasma.

The effect of old age on the disposition and antinociceptive response of morphine and morphine-6β-glucuronide in the rat

Abstract The aims of this study were to examine the effect of old age on the pharmacokinetics of morphine and morphine‐6&bgr;‐glucuronide (M6G) and their relationships to antinociceptive activity. Morphine (21.0 &mgr;mol/kg) or M6G (21.7 &mgr;mol/kg) were administered s.c. to young adult and aged male Hooded‐Wistar rats. Antinociceptive effect was measured by the tail‐flick method at various times up to 2.5 h or 6.5 h after morphine or M6G administration, respectively, and concentrations of morphine, morphine‐3&bgr;‐glucuronide (M3G) and M6G in plasma and brain were determined by HPLC. Creatinine clearance was significantly lower by 33% or 21% in aged compared to young adult rats receiving morphine or M6G, respectively. After morphine administration, the areas under the (i) antinociceptive effect‐time curve, (ii) plasma morphine concentration‐time curve, and (iii) brain morphine concentration‐time curve were not different between young adult and aged rats. However, the AUC for plasma M3G was five‐fold higher in the aged relative to young adult rats, which could not be accounted for by only a 33% lower creatinine clearance. M6G was not detected in any plasma or brain sample from rats administered morphine and no M3G was detected in brain. For M6G administration, the areas under the (i) antinociceptive effect‐time curve, and (ii) plasma M6G concentration‐time curve were 1.8‐ and 1.6‐fold higher in aged compared to young adult rats, respectively. Concentrations of M6G in brain were below the limit of quantification. No morphine or M3G was detected in any of the plasma or brain samples of rats administered M6G. The results demonstrate no change in morphine antinociception and pharmacokinetics with age, and suggest that blood‐brain barrier permeability and receptor sensitivity to morphine are not altered in aged rats. Accumulation of M3G in plasma of aged rats is probably due to diminished renal clearance of M3G in addition to a reduction in the biliary excretion of M3G. The heightened sensitivity of the aged rats to M6G is probably due to the observed altered kinetics of M6G rather than a pharmacodynamic change.

Stereoselective interactions of ketoprofen glucuronides with human plasma protein and serum albumin

A clearance pathway common to many aryl alkanoic acids is the generation of renally eliminated ester glucuronides. These metabolites are susceptible to systemic hydrolysis which generates the parent aglycone. We have conducted in vitro studies with biosynthetic R- and S-ketoprofen glucuronides to elucidate the mechanism of this phenomenon. These conjugates were incubated in human plasma, various concentrations of human serum albumin (HSA) and protein-free buffer. It was apparent that albumin, rather than plasma esterases, catalysed the hydrolysis of the glucuronides. The albumin-catalysed hydrolysis of ketoprofen glucuronides was highly stereoselective. The mean (+/- SD) hydrolysis half-life of R-ketoprofen glucuronide in plasma (N = 4) at physiological pH and temperature was 1.37 (+/- 0.30) hr. The corresponding value for S-ketoprofen glucuronide, 3.46 (+/- 0.84) hr, was significantly different (P less than 0.005). In contrast, synthetic ethyl esters of R- and S-ketoprofen were hydrolysed by plasma esterases, but not by HSA, and with little stereoselectivity. The reversible protein binding of ketoprofen glucuronides was determined at physiological pH and temperature by a rapid ultra-filtration method. The binding of R- and S-ketoprofen glucuronide to human plasma protein was independent of concentration (P greater than 0.05) over the range of 1-20 micrograms/mL. The mean (+/- SD) percentage unbound in plasma (N = 4) of R-ketoprofen glucuronide was 12.6 (+/- 1.4)%. The corresponding value for S-ketoprofen glucuronide, 9.12 (+/- 0.54)%, was significantly different (P less than 0.005). S-Ketoprofen glucuronide was also more avidly protein bound in physiological concentrations of HSA. However, this stereoselectivity decreased in more dilute HSA solutions. Based on the hydrolysis and protein binding data for ketoprofen glucuronides, we propose the existence of separate binding and catalytic sites on the albumin molecule for these metabolites.

Glucuronidation of dihydrocodeine by human liver microsomes and the effect of inhibitors.

1. Glucuronidation is the major route of metabolism of dihydrocodeine (DHC) and accounts for 25–30% of an oral dose in urine. The kinetics of DHC‐6‐glucuronide formation in liver microsomes from five human donors and the effect of a number of potential inhibitor drugs were examined using a newly developed and validated HPLC assay.

CONCENTRATION‐EFFECT RELATIONSHIPS OF MORPHINE AND MORPHINE‐6β‐GLUCURONIDE IN THE RAT

1. The aims of the present study were to determine the relationship between the antinociceptive effect and concentrations of morphine and morphine‐6β‐glucuronide (M6G) in plasma and in the brain.