Kenneth F. Brown

Displacement of tolbutamide, glibenclamide and chlorpropamide from serum albumin by anionic drugs☆

Abstract The binding of tolbutamide, chlorproramide and glibenclamide to human and bovine serum albumin has been estimated in the presence of a number of acidic drugs. It is shown that agreement between experimental data and that calculated using the competitive binding equation is very poor. The degree of displacement of tolbutamide and chlorpropamide is much greater than that calculated using the equation while displacement of glibenclamide is much less. These findings suggest that displacement is essentially non-competitive and that glibenclamide is less susceptible to displacement by acidic drugs than tolbutamide or chlorpropamide.

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.

The binding of sulphonylureas to serum albumin

The interaction of tolbutamide, glibenclamide, chlorpropamide and tolazamide with serum albumin has been examined. Glibenclamide, the most strongly bound of the four compounds, is bound to only one class of sites. The other three compounds are bound to at least two. The interaction between glibenclamide and albumin was independent of pH and increased markedly with decreasing temperature suggesting that a non‐ionic mechanism is involved. In contrast, the overall interaction of tolbutamide with albumin showed little temperature dependence and, in addition, binding of both tolbutamide and chlorpropamide decreased with pH. These findings imply that the predominantly bound species is the anion. Binding parameters corrected for electrostatic effects were found to fit binding data for tolbutamide, chlorpropamide and tolazamide better than uncorrected parameters. Electrostatic correction of binding of glibenclamide is unnecessary.

Cyclosporine plasma unbound fraction in heart and lung transplantation recipients.

To investigate the variability in the unbound fraction (fu) of cyclosporine in recipients of heart, heart-lung, and lung transplantation, cyclosporine fu was determined ex vivo in plasma by equilibrium dialysis. In a retrospective study, 260 samples of plasma (one to seven per patient) were obtained from 89 heart (86%), lung (9%), and heart-lung (5%) transplant patients. The unbound fraction (x100) of cyclosporine ranged from 0.52% to 3.94%, with an overall mean of 1.53%+/-0.375% (SD). The mean percentage unbound for individual patients ranged from 0.71% to 1.98%, giving a 2.8-fold interpatient variation. In heart transplant recipients (66 patients), the values of fu were significantly lower (p < 0.01) during more severe rejection episodes, which required antirejection treatment (endomycardial biopsy result of grade 3a and higher) than in the absence of rejection (grade 0) or during grade la rejections. The value of fu did not vary with organ transplanted (p = 0.35) or etiology of organ failure (p = 0.32). Cyclosporine fu was negatively correlated with the age of the patient (r = -0.18, p < 0.05). Correlations were not observed between fu and blood biochemical and cytologic indices. However, fu was significantly lower (p < 0.01) in hypercholesterolemic transplant recipients (1.37+/-0.52%) than in normocholesterolemic patients (1.60+/-0.63%). Administration of simvastatin resulted in a significant increase in the mean fu from 1.40+/-0.09%) to 1.82+/-0.13% (paired t test, n = 13; p < 0.01). In patients who received ketoconazole, fu was not different from controls. These findings suggest that the level of cyclosporine fu may be an important determinant of immunosuppressive activity of cyclosporine. Moreover, the variation in fu could be strongly related to the concentration of serum lipoproteins; interpretation of the results of cyclosporine monitoring thus requires consideration of the lipidemic status of the patient.

Transsynovial distribution of ibuprofen in arthritic patients

Free and total ibuprofen levels in serum and synovial fluid (SF) were determined in one male and 14 female arthritic patients (mean age 56 yr, range 19 to 77) after 400 mg three times daily for 2 days. Free drug was separated by ultrafiltration and assayed by a new highly sensitive and precise gas chromatographic–mass spectrometric technique. Total protein and albumin were assayed by automatic analyzer, and the routine serum biochemical profile obtained. Serum and S F none sterified fatty acids (NEFAs) were methylated and assayed by gas chromatography. While both total protein and albumin levels were lower (p < 0.01) in SF, NEFA levels did not differ (p > 0.05). Ibuprofen readily partitions into and binds strongly to joint fluid, the total level being about half that in serum. The ratio of total ibuprof en in SF to that in serum (x̄ = 0.41 ± 0.17) correlated with the albumin concentration ratio (r = 0.886) indicating that albumin binding is an important determinant of ibuprof en transsynovial distribution. A low total ibuprofen SF/serum ratio, apparent for three patients on concomitant aspirin therapy, is consistent with reports of an ibuprofen‐salicylate interaction. Free ibuprofen in SF (0.192 ± 0.086 µg ml−1) did not differ (p > 0.05) from serum free levels (x̄ = 0.251 ± 0.139 µg ml−1), supporting the concept of the synovial cavity as a compartment readily accessible to unbound drug species. Multiple regression analyses revealed relative independence of free concentration on total levels, and the small positive and negative influences of NEFAs and albumin concentrations. Bilirubin, uric acid, cholesterol, triglycerides, and chloride ion did not correlate with serum free concentration.

High-performance liquid chromatographic analysis of mometasone furoate and its degradation products: application to in vitro degradation studies.

A method of analysis of mometasone furoate in pharmaceutical formulations and biological fluids is necessary to study the degradation kinetics and determine its stability. A simple high-performance liquid chromatographic method was developed for simultaneous determination of mometasone furoate and its degradation products in human plasma. Plasma (0.5 ml) was extracted with dichloromethane after addition of the internal standard, dexamethasone 21-acetate. Separation was achieved on a Beckman C(8) column with UV detection at 248 nm. The calibration curve was linear ranging from 0.2 to 100 microg/ml. The mean extraction efficiency was >86%. Precision of the assay was <10% (CV), and was within 10% at the limit of quantitation (0.2 microg/ml). Bias of the assay was lower than 7%. The limit of detection was 50 ng/ml for a 0.5-ml sample. The assay was applied successfully to the in vitro kinetic study of degradation of mometasone furoate in human plasma and simulated biological fluids.

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.

Determination of epimers 22R and 22S of budesonide in human plasma by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A highly sensitive and selective method has been developed for the simultaneous quantification of 22R- and 22S-epimers of budesonide in human plasma. The drug was isolated from human plasma using C18 solid-phase extraction cartridges and was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives. Deuterium-labelled budesonide was synthesized and determined to have an isotopic purity > 99%. This was used as the internal standard. Epimers were quantified by automated liquid chromatography-atmospheric pressure chemical ionization mass spectrometry, operating in selected ion mode at m/z 473.2 and m/z 476.2. Linear responses were observed for both epimers over the range 0.25 to 10.0 ng/ml. The average recoveries of 22R- and 22S-epimers of budesonide from human plasma were 87.4% and 87.0%, respectively. The lower limit of quantification for each epimer was 0.25 ng/ml, corresponding to 50.0 pg of analyte on column. Within- and between-day coefficients of variation were 8.6% and 4.0%, respectively.

Comparative Kinetics of Metabolism of Beclomethasone Propionate Esters in Human Lung Homogenates and Plasma

The systemic availability of inhaled beclomethasone dipropionate (BDP) is the net result of the absorption of the glucocorticoid from the lower respiratory and gastrointestinal tracts, and metabolism in the lung, plasma, and other sites. The metabolism kinetics of BDP and its active metabolite, beclomethasone 17-monopropionate (17-BMP), in human lung 1000 x g supernatant (HLu) and human plasma (HP) at 37 degrees C were compared. The effect of MgCl(2) and/or an NADPH-generating system on the decomposition of BDP and 17-BMP in HLu was also investigated. The concentrations of BDP and its metabolites were determined by HPLC with UV detection at 242 nm. Kinetics of decomposition of BDP and 17-BMP in HLu and HP were qualitatively and quantitatively different. The decomposition of BDP in HLu involved only hydrolysis. In comparison, three reactions are involved following incubation of BDP in HP; namely, hydrolysis, transesterification, and loss of hydrogen chloride. The hydrolysis of BDP and 17-BMP in HLu seem to be inhibited appreciably by MgCl(2) with the NADPH-generating system. Effective activation of BDP in HLu, in combination with transesterification of 17-BMP in HP, might favor a high ratio of local antiinflammatory activity to systemic side effects following inhalation of BDP.

UNBOUND CYCLOSPORINE AND ALLOGRAFT REJECTION AFTER HEART TRANSPLANTATION

BACKGROUND To determine the impact of cyclosporine plasma protein binding on organ rejection after cardiac transplantation, the incidence of cardiac rejection episodes was compared among patients who had differing levels of cyclosporine plasma fraction unbound (fU). METHODS Forty-six consecutive cardiac transplant recipients were sampled at 1, 3, 6, and 12 months after transplantation, and cyclosporine plasma fU was determined, using a specially developed equilibrium dialysis method. At the completion of the study, incidences of cardiac rejection episodes were compared among patients having mean cyclosporine fU (Csfu) that were low (LCsfu; mean+/-SD, 1.33+/-0.10%, n=15), intermediate (ICsfu; 1.60+/-0.07%, n= 16), and high (HCsfu; 1.99+/-0.30%, n=15). RESULTS Percentage of endomyocardial biopsies (grade 3a, 3b, and 4) with respect to the total number of biopsies performed in the first 3 months after transplant was significantly higher in the LCsfu group than the other groups (40.9% in LCsfu vs. 28.5% for ICsfu and 32.1% for HCsfu groups, P=0.02). The linearized rate of rejection (episodes of rejection/100 patient-days) in the first month after transplant was 6.5+/-1.7 for LCsfu, 3.5+/-0.8 for ICsfu and 4.3+/-0.9 for the HCsfu group (P<0.05, low vs. intermediate-high). The mean (95% confidence interval) of time interval between the first and second episodes of rejections was 10.7 (5.6-16.0) days for LCsfu, 18.0 (8.6-29.0) days for the ICsfu, and 26.0 (15.1-36.9) days for the HCsfu group (P<0.01). The total number of rejections requiring treatment per patient in the first 3 months after transplant was higher in the LCsfu group compared with the others (4.0+/-1.7 episodes for LCsfu vs. 2.9+/-1.1 for ICsfu and 3.2+/-1.2 episodes for HCsfu; P<0.05). Four patients in the low group, one patient in the intermediate group, and no patients in the high group required treatment with total lymphoid irradiation (P<0.02). CONCLUSIONS This finding suggests that patients with lower levels of cyclosporine fU are more prone to cardiac rejection and that the level of cyclosporine fraction unbound may be clinically important for determination of response to cyclosporine therapy.