Validation of methods to study the distribution and protein binding of tacrolimus in human blood.

INTRODUCTION: Tacrolimus is a macrolide immunosuppressant that has a narrow therapeutic index, displays considerable variability in response, and has the potential for serious drug interactions. Therapeutic drug monitoring and dose individualisation for tacrolimus is complicated but essential. Few studies have investigated the blood distribution and protein binding of tacrolimus and the results of these studies are conflicting. The aim of the present study is to establish and validate methods to investigate the distribution of tacrolimus in human blood. To conduct these studies at clinically relevant concentrations the use of 3H-dihydro-tacrolimus instead of tacrolimus was investigated. METHODS: The use of radiolabelled tacrolimus was validated by conducting studies with a mixture of both labelled and unlabelled drug where tacrolimus was analysed by LC-MS/MS. The in vitro distribution of tacrolimus and 3H-dihydro-tacrolimus was investigated in blood collected from healthy subjects using Ficoll-Paque reagent and density gradient ultracentrifugation, respectively. The unbound fraction of tacrolimus in plasma was studied using equilibrium dialysis conducted at 37 degrees C. RESULTS: In blood, tacrolimus was found to be mainly associated with erythrocytes (85.3+/-1.5%), followed by diluted plasma proteins (14.3+/-1.5%) and lymphocytes (0.46+/-0.10%). In plasma, tacrolimus was found to mainly be associated with the soluble protein fraction (61.2+/-2.5%), high-density lipoproteins (HDL, 28.1+/-5.4%), low-density lipoproteins (LDL, 7.8+/-1.6%), and very low-density lipoproteins (VLDL, 1.4+/-0.3%). The unbound fraction of tacrolimus was found to be only 1.2+/-0.12%. Statistical comparison indicated that there was no significant difference in the blood distribution and plasma protein binding of 3H-dihydro-tacrolimus when compared with tacrolimus. DISCUSSION: These results have important implications for therapeutic drug monitoring of tacrolimus and subsequent studies of tacrolimus distribution in transplant recipients.

Photodegradation of amiloride in aqueous solution.

The photodegradation of amiloride hydrochloride in deaerated aqueous solution at 30 degrees C in the pH range 4.5-11.0 was studied by spectrophotometry and reversed-phase HPLC. The neutral form of the drug present in alkaline solution degraded approximately 3-fold faster than the cationic form. The quantum yields for photodegradation of neutral amiloride and its conjugate acid were determined using ferrioxalate actinometry to be 0.023+/-0.002 and 0. 009+/-0.001, respectively. The initial photoreaction involves dechlorination of amiloride and the major product is N-amidino-3, 5-diamino-6-hydroxylpyrazine-carboxamide, established by UV, 1H and 13C NMR, and chemical ionization-mass spectrometry. The mechanism of photolysis is postulated to involve cation radical formation that facilitates the dechlorination step. The photosensitizing activity of amiloride hydrochloride was tested by measuring (a) the rate of oxygen uptake in the presence of singlet oxygen substrates, 2, 5-dimethylfuran or l-histidine, and (b) the rate of free radical polymerization of acrylamide, at 30 degrees C in aqueous solution. Photosensitization by amiloride was concluded to occur predominantly via singlet oxygen rather than a free radical mechanism. However, amiloride is a much weaker photosensitizer than other diuretics such as frusemide and hydrochlorothiazide, tested under the same experimental conditions.

Degradation products of beclomethasone dipropionate in human plasma.

The anti-inflammatory glucocorticosteroid beclomethasone dipropionate was found previously to degrade in human plasma at 37 degrees C to yield beclomethasone 17-monopropionate, beclomethasone 21-monopropionate, and beclomethasone together with three unknown species, D-1, D-2, and D-3. In this paper, we report the isolation of D-2 and D-3 by preparative HPLC and the elucidation of their structures. Both products D-2 and D-3 exhibited UV bathochromic shifts relative to beclomethasone dipropionate of 9 nm. From the mass spectrometry and 1H-NMR data, it is concluded that D-2 and D-3 are formed from beclomethasone and beclomethasone 21-monopropionate, respectively, with the loss of hydrogen chloride and the formation of a 9,11-epoxide. Data for 1H-NMR methyl chemical shifts are used to show that the epoxide has the mechanistically more plausible 9beta,11beta configuration. Thus, D-2 is 9beta, 11beta-epoxy-16beta-methyl-1,4-pregnadiene-17alpha,21- diol-3, 20-dione, and D-3 is its corresponding 21-propanoate. The various enzyme-catalyzed and nonenzyme-catalyzed reactions involved in the degradation of beclomethasone dipropionate in human plasma are discussed. A degradation scheme is proposed.

Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria.

The pathogenesis of human cerebral malaria (CM) remains unresolved. In the most widely used murine model of CM, the presence of T lymphocytes and/or interferon (IFN)-gamma is a prerequisite. IFN-gamma is the key inducer of indoleamine 2,3-dioxygenase (IDO), which is the catalyst of the first, and rate-limiting, step in the metabolism of tryptophan (Trp) along the kynurenine (Kyn) pathway. Quinolinic acid (QA), a product of this pathway, is a neuro-excitotoxin, like glutamic acid (Glu) and aspartic acid (Asp). Kynurenic acid (KA), also produced from the Kyn pathway, antagonizes the neuro-excitotoxic effects of QA, Glu, and Asp. We therefore examined the possible roles of IDO, metabolites of the Kyn pathway, Glu, and Asp in the pathogenesis of fatal murine CM. Plasmodium berghei ANKA infection was studied on days 6 and 7 post-inoculation (p.i.), at which time the mice exhibited cerebral symptoms such as convulsions, ataxia, coma, and a positive Wooly/White sign and died within 24 hours. A model for noncerebral malaria (NCM), P. berghei K173 infection, was also studied on days 6 and 7 and 13 to 17 p.i. to examine whether any changes were a general response to malaria infection. Biochemical analyses were done by high-pressure liquid chromatography and gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS). IDO activity was low or absent in the brains of uninfected mice and NCM mice (days 6 and 7 p.i.) and was induced strongly in the brains of fatal murine CM mice (days 6 and 7 p.i.) and NCM animals (days 13 to 17 p.i.). This induction was inhibited greatly by administration of dexamethasone, a treatment that also prevented CM symptoms and death. Furthermore, IDO induction was absent in IFN-gamma gene knockout mice, which were also resistant to CM. Brain concentrations of Kyn, 3-hydroxykynurenine, and the neuro-excitotoxin QA were significantly increased in both CM mice on days 6 and 7 p.i. and NCM mice on days 13 to 17 p.i., whereas an increase in the ratio of brain QA to KA occurred only in the CM mice at the time they were exhibiting cerebral symptoms. Brain concentrations of Glu and Asp were significantly decreased in CM and NCM mice (days 13 to 17 p.i.). The results imply that neuro-excitation induced by QA may contribute to the convulsions and neuro-excitatory signs observed in CM.

A sensitive method for the quantification of fluticasone propionate in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry.

A highly sensitive and selective method has been developed for the quantification of fluticasone propionate (FP) in human plasma. The drug was isolated from human plasma using C18 solid-phase extraction cartridges. The analysis was based on high-performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry (HPLC/APCI/MS), using the 22R epimer of budesonide (BUD) acetate, synthesised using acetic anhydride, as internal standard. The mass spectrometer was operated in APCI mode with selected ions at tune masses of 473.2 and 501.2 m/z, corresponding to the MH+ of acetylated (22R)BUD and FP, respectively. The mobile phase used was a mixture of 50% ethanol in water with a flow rate of 0.45 ml min-1. The system was optimised by tuning the capillary and tube lens with a concentrated solution of FP. The recovery of FP from human plasma was 86.3%. Linearity of response was obtained over the concentration range 0.2-4.0 ng ml-1. The intra-assay and inter-assay variability were 6.3 and 2.9%, respectively. The lower limit of quantification was 0.2 ng ml-1 when a solid-phase extraction preceded the HPLC/APCI/MS.