Combined oral cyclosporin and methotrexate therapy in patients with rheumatoid arthritis elevates methotrexate levels and reduces 7-hydroxymethotrexate levels when compared with methotrexate alone.

OBJECTIVE: To study the pharmacokinetics of methotrexate (MTX) plus cyclosporin A (CSA) in patients with rheumatoid arthritis (RA). METHODS: On day 1 of the study, patients with RA receiving stable doses of MTX had blood and urine levels of MTX and its metabolite 7-hydroxymethotrexate (7-OH-MTX) measured post oral dosing of the drug. MTX was then discontinued and CSA therapy was started on day 8. On day 20, blood levels of CSA and CSA metabolites were measured post drug dosing. On day 23, MTX therapy was restarted and levels of MTX, CSA and their metabolites were again measured as described above. RESULTS: In the 30 patients, coadministration of CSA and MTX led to a 26% increase in mean peak plasma MTX concentration (P < 0.01), an 18% increase in the mean plasma MTX concentration area under the curve (AUC, P=0.01) and an 80% decrease in plasma 7-OH-MTX AUC (P < 0.01). In 13 patients receiving a 10 mg MTX dose, CSA reduced urinary 7-OH-MTX excretion by 87% (P < 0.01) without altering MTX excretion. MTX did not alter the pharmacokinetics of CSA or its metabolites. CONCLUSION: CSA may block oxidation of MTX to its relatively inactive metabolite, 7-OH-MTX, thereby potentiating MTX efficacy.

Improved cyclosporine bioavailability in black pediatric liver transplant recipients after administration of the microemulsion formulation.

Black transplant recipients are associated with low cyclosporine bioavailability, which may contribute to the poorer clinical outcomes observed with these patients. In this analysis, we compared cyclosporine exposure in black (n = 9) and nonblack (n = 18) pediatric maintenance liver transplant recipients by using steady-state pharmacokinetic profiles obtained after administration of the original and microemulsion formulations of cyclosporine. Treatment with the original cyclosporine formulation resulted in lower mean dose-normalized, area under the concentration-versus-time curve values for black compared with nonblack pediatric liver transplant recipients. On conversion to the microemulsion formulation of cyclosporine, black and nonblack patients experienced increases in cyclosporine bioavailability of 102% and 39%, respectively (P =.009 and P =.001). Because the increase in mean bioavailability was substantially greater for blacks, area under the concentration-versus-time curve values for this pediatric subpopulation became similar to those levels obtained for nonblacks receiving the microemulsion formulation for cyclosporine. When patients were further stratified by age, ethnic differences in bioavailability with the original formulation of cyclosporine were most apparent in the 1- to 5-year age group. Conversion to the microemulsion formulation resulted in a 164% increase (P =.05) in bioavailability for black patients within this age group such that, again, these levels became similar to area under the concentration-versus-time curve values obtained for young nonblacks receiving cyclosporine for microemulsion. Improvements in cyclosporine bioavailability after administration of the microemulsion formulation of cyclosporine may translate to improved long-term graft and patient outcomes for black pediatric liver transplant recipients.

Equivalence of cyclosporine blood level assays in patients receiving cyclosporine microemulsion or cyclosporine. The Sandoz Neoral Study Group N103.

The more rapid absorption of the cyclosporin A (CyA) microemulsion formulation (Neoral, NEO) compared to Sandimmune (SIM) might bypass intestinal metabolism resulting in differing amounts of CyA metabolites in blood as compared to SIM. If true, then CyA levels obtained with a CyA monoclonal antibody assay (TDx) that has metabolite cross-reactivity might differ depending on the CyA formulation received by the patient, thereby affecting safety and efficacy. Fifty-one NEO vs. 50 SIM treated de novo renal transplant recipients from a multicenter double-blind randomized trial had morning, whole-blood, trough-samples obtained at the ends of weeks 1, 4, 8, and 12 post-transplant assayed for CyA by HPLC and TDx. The slopes (ratio of TDx value to HPLC value) for the regression lines between TDx and HPLC levels as a function of time post-transplant and CyA formulation were determined using a general linear model. For NEO, the slopes at each week (1.21-1.41 x HPLC) did not differ significantly (p = 0.82). For SIM, the week 1 slope (1.2) was significantly (p = 0.006) less than the other weeks (1.4-1.44). The slopes (NEO vs. SIM) were not different at either week 1 (1.21 vs. 1.22, p = 0.82) or at pooled weeks 4, 8, and 12 (1.33 vs. 1.4, p = 0.1). These results indicate that despite the improved absorption, TDx values obtained on NEO are qualitatively similar to those obtained on SIM.

Pharmacokinetics of an oral solution of the microemulsion formulation of cyclosporine in maintenance pediatric liver transplant recipients.

BACKGROUND: A comparison of the oral bioavailability of cyclosporine from the original formulation (CsA) and from the new formulation, cyclosporine for microemulsion (CsA-ME), was made in pediatric maintenance liver transplant patients within two age groups (group 1, ages 1-5 years; group 2, ages 6-17 years) in an open-label, multicenter, randomized crossover trial. All patients were at least 6 months past transplantation and were receiving CsA maintenance therapy. METHODS: In study period 1 (days 1 through 14), patients were administered either CsA or CsA-ME at the same b.i.d. dosage as their maintenance therapy. Upon entry into period 2 (days 15 through 28), patients were converted to the alternate formulation at a 1:1 mg dose ratio. On day 29, all patients returned to the CsA treatment administered at study entry, with follow-up on day 35. Dosage adjustments were not allowed with either CsA or CsA-ME. Twelve-hour pharmacokinetic profiling was performed at the end of periods 1 and 2. RESULTS: Both the mean area under the concentration-versus-time curve and the mean maximum blood concentration of cyclosporine-both normalized for dose-were significantly increased: by 66% and 109%, respectively, in patients receiving CsA-ME compared with those receiving CsA in group 1 and by 39% and 75%, respectively, in group 2. During this study, liver function remained stable, and serum creatinine and blood pressure did not differ significantly between treatment groups. CONCLUSIONS: This study shows increased bioavailability in all patients converted to CsA-ME, with the greatest increase seen in patients with the lowest initial cyclosporine bioavailability. The tolerability was similar between the two formulations during this study.

Safety and tolerability of cyclosporine microemulsion versus cyclosporine: two-year data in primary renal allograft recipients: a report of the Neoral Study Group.

BACKGROUND: The new microemulsion formulation of cyclosporine (CsA-ME) is more bioavailable than cyclosporine (CsA) in de novo renal transplant patients. Therefore, it was of interest to compare the safety profile of each formulation in such patients. METHODS: In a multicenter, double-blind, parallel-group study, 101 renal transplant recipients were randomized after transplantation to receive either CsA (n=50) or CsA-ME (n=51) capsules twice daily for 2 years. Of these patients, 54 (CsA, n=26; CsA-ME, n=28) completed 1 year of the study and entered the second-year, double-blind extension. Initial dose at the time of transplantation was 5 mg/kg b.i.d.; doses were titrated to target trough levels. METHODS: The mean (+/- SD) doses at the end of 2 years were 4.6 +/- 1.8 and 3.8 +/- 1.1 mg/kg per day for CsA- and CsA-ME-treated patients, respectively. The mean (+/- SD) CsA trough levels at end point were 187 +/- 63 and 210 +/- 95 ng/ml for CsA- and CsA-ME-treated patients, respectively. At least one adverse event was reported by 25/26 (96%) of CsA- and 27/28 (96%) of CsA-ME-treated patients. No patient discontinued the study because of adverse events. No deaths occurred during the study. Renal function, as measured by serum creatinine levels, and blood pressure were comparable over time in both treatment groups. CONCLUSIONS: There was no significant difference in safety and tolerability between CsA- and CsA-ME-treated kidney recipients for 2 years after transplantation.

The pharmacokinetics of a microemulsion formulation of cyclosporine in primary renal allograft recipients. The Neoral Study Group.

This study was a randomized, double-blind, 12-week comparison of the pharmacokinetics, safety, and tolerability of two cyclosporine (CsA) formulations, cyclosporine emulsion capsules and oral solution for microemulsion and cyclosporine, in the postoperative management of renal transplant patients. Of the 101 patients, aged 18 to 65, who entered the study, 89 were evaluable for pharmacokinetics. Initial dosage was 10 mg/kg per day, administered twice daily in two equal doses. Dosages were adjusted to achieve target CsA concentrations. The pharmacokinetic (PK) parameters (dose-normalized) of greatest interest were maximum blood concentration (C(max)/dose), time to reach maximum concentration (t(max), area under the blood concentration-vs.-time curve (AUC/dose), and trough blood concentrations (Co h/dose). The relative CsA bioavailabilty was found to be significantly enhanced with cyclosporine emulsion compared with cyclosporine with a 16% to 31% increase in AUC and a 32% to 42% increase in C(max). Intrapatient variability of PK parameters was significantly lower with cyclosporine emulsion than with cyclosporine for AUC, C(oh), t(max), and C(max) in many instances. This indicates a more consistent, rapid, and more complete total absorption of CsA. Despite higher CsA C(max) levels and AUCs with cyclosporine emulsion, safety and tolerability (detailed in a parallel report) were comparable to those of cyclosporine. The PK advantages of cyclosporine emulsion over cyclosporine are either independent of food conditions or possibly reflective of more consistent absorption of CsA with cyclosporine emulsion. The findings suggest that de novo use of cyclosporine emulsion may simplify and improve management of organ transplant recipients and that the PK advantages of cyclosporine emulsion may translate into clinical benefits.

The safety and tolerability of cyclosporine emulsion versus cyclosporine in a randomized, double-blind comparison in primary renal allograft recipients. The Neoral Study Group.

A 12-week, randomized, double-blind, multicenter pharmacokinetics study was conducted to compare the clinical safety and tolerability of cyclosporine capsules and oral solution for microemulsion and cyclosporine in 101 primary renal transplant recipients Cyclosporine emulsion has more complete absorption and improved bioavailability compared with cyclosporine, and dosing of both cyclosporine formulations was adjusted to achieve comparable whole-blood trough levels. Mean serum creatinine values were higher in the cyclosporine emulsion group at baseline, 8, and 12 weeks (P<0.05). The incidence of acute rejection was similar in both treatment groups although fewer patients required monoclonal antibody therapy in the cyclosporine group (31% vs. 82%, respectively). Despite the increased bioavailability of cyclosporine emulsion, no significant differences in the incidence of adverse events were observed; the safety, tolerability, and efficacy of cyclosporine emulsion and cyclosporine were comparable.

Application issues in bioanalytical method validation, sample analysis and data reporting.

Although some degree of consensus has been reached concerning the requirements for acceptable method validation, the procedures used to establish them vary significantly between laboratories. Also, issues arising from application of these requirements during validation and subsequent sample analysis need to be addressed. The purpose of this paper is to discuss application issues concerning prerequisites to method validation, and all validation criteria for evaluation of method reliability and overall performance. Other poorly addressed issues such as re-validation, cross-validation, partial sample volume, multicomponent analysis and reporting will also be discussed. Although many issues discussed are of a general nature, the scope of this presentation is primarily to address issues arising from the validation and routine application of chromatographic methods.

High-performance liquid chromatographic method for the determination of fluvastatin in human plasma.

The method outlined in this paper utilizes internal standardization and is simple, reliable, and sensitive for the determination of fluvastatin in plasma. Fluvastatin sodium and the internal standard are extracted from buffered plasma into methyl tert.-butyl ether, followed by evaporation of an aliquot of the organic phase. After reconstitution of the dried sample into a small volume of mobile phase (methanol-13 mM tetrabutylammonium fluoride, 3:2, v/v), the sample is chromatographed on an LC-18 column thermostated at 50 degrees C. Fluorescence detection (excitation at 305 nm and emission at 380 nm) is used to monitor both fluvastatin (free acid) and the internal standard. The method can accurately detect 1 ng/ml fluvastatin using a 1.0-ml plasma sample. The precision and reproducibility over the linear range of the method are 5.57 and 7.32%, respectively. This method has been used to measure fluvastatin plasma concentrations in support of bioavailability/pharmacokinetic studies with no indication of interference.

Infrared spectra of carbonyl hemoglobins: characterization of dynamic heme pocket conformers.

The infrared spectra for carbon monoxide complexed to hemoglobins were examined in the C-O stretch region. Deconvolution of the spectra requires four bands and supports the presence of four distinct conformers at the ligand binding site. Most typical hemoglobins exhibit only one predominant conformer for each subunit represented by a band at 1951 cm-1 in contrast to myoglobins, which typically exist in two major conformations. Several hemoglobins with an enlarged heme pocket are shown to shift the C-O frequency into the higher frequency conformer regions. Many factors, including pH, temperature, solvents, and divalent metals, are also shown to be capable of expanding the heme pocket. Only very specific structural changes that can reduce the size of the heme pocket will result in the lower frequency conformers. The weighted averages of the multiple CO vibrational frequencies are linearly related to the single 13CO NMR chemical shift values and to the exponential of fast CO on-rates. Conformer interconversion occurs at a rate greater than 10(4) s-1. The infrared C-O stretch spectra provide qualitative and quantitative information on the structural dynamics, stability, and ligand binding properties of hemoglobins.

Single- vs multiple-dose pharmacokinetics of clozapine in psychiatric patients.

Clozapine plasma levels were monitored in 16 patients during a series of three consecutive treatments (single dose-multiple dose-single dose). Each patient received a single 75-mg dose (3 x 25 mg) with clozapine tablets, and serial plasma samples were collected over 48 hr after the dose. At 48 hr, a multiple-dose regimen was started, consisting of an initial dose escalation period followed by dosing at a constant regimen for at least 6 days. After the last dose, serial plasma samples were again obtained over 72 hr. Drug was then withheld for at least 7 days, a final single 75-mg dose was given, and plasma sampling was repeated. A subset of the patient population (N = 7) was used to test for a food effect during the single-dose treatments. The pharmacokinetic parameters between the initial and the final single dose periods were not significantly different. Similarly, there were no differences within patients when given the dose after fasting (fed 1 hr after dose) or with a meal. In contrast, the terminal elimination rate differed between the single-dose and the multiple-dose treatments (t1/2 m3 = 7.9 hr single dose and 14.2 hr multiple dose) (P less than 0.05) and the dose-normalized area under the plasma concentration/time curves increased 27% with multiple dosing. Since a previous study in patients (Choc et al., Pharm. Res. 4:402-405, 1987) showed dose proportionality of clozapine plasma concentrations during multiple-dose regimens, the present results cannot be described by Michaelis-Menten kinetics.

Single dose kinetics of thioridazine and its two psychoactive metabolites in healthy humans: a dose proportionality study.

Dose proportionality in some pharmacokinetic parameters for thioridazine and its two active metabolites (mesoridazine and sulforidazine) was investigated in 11 healthy human subjects following oral administration of three single doses (25, 50, and 100 mg) of thioridazine hydrochloride separated in each case by an interval of two weeks. Also, after a further two weeks, another 100-mg dose of thioridazine (divided as 5 mg every 0.5 h) was administered to all the volunteers to investigate the effect of a slow rate of dosage input on the pharmacokinetic parameters of this drug. An HPLC method was used to measure concentrations of thioridazine, mesoridazine, and sulforidazine in plasma samples collected up to 72 h following each dose. Dose proportionality for the three single doses of thioridazine was observed for all three analytes in the area under the plasma concentration versus time curves (AUC infinity 0 or AUCt0) and the maximum plasma concentration (Cmax) in that the relationships between the dose and these parameters were each describable by an equation for a straight line (r2 greater than or equal to 0.8). However, the mean apparent distribution and elimination rate constants for thioridazine and mesoridazine and the mean apparent oral clearance for thioridazine decreased significantly with increasing dose. This suggests nonlinear trends in the elimination kinetics at high doses of thioridazine. When a 100-mg divided oral dose of thioridazine was administered, no statistically significant differences between single and divided doses were observed in the mean AUC infinity 0 or AUCt0 for thioridazine or sulforidazine. A significant decrease in the mean AUC infinity 0 or AUCt0 was observed for mesoridazine after the administration of the divided dose.(ABSTRACT TRUNCATED AT 250 WORDS)

The site and mechanism of dioxygen reduction in bovine heart cytochrome c oxidase.

The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.

S-oxidation of thioridazine to psychoactive metabolites: an oral dose-proportionality study in healthy volunteers.

Thioridazine has two major active metabolites, which are formed from S-oxidation of its 2-methylthio group; the sulphoxide, mesoridazine, and the sulphone, sulforidazine. Dose proportionality of the three compounds was investigated for the first time in 11 males after administration of three single oral doses (25, 50, and 100 mg) of thioridazine hydrochloride separated in each case by two weeks. Based on the plasma concentrations of the three analytes over 72 h following each dose, large intersubject variabilities in such parameters as AUCot and Cmax were observed for each of the three compounds. The relationships between dose and parameters such as AUCot and Cmax for each analyte were described by an equation for a straight line (r2 greater than or equal to 0.8). However, the mean apparent distribution and elimination rate constants for thioridazine and mesoridazine and the mean apparent oral clearance for thioridazine decreased significantly with increasing dose, suggesting non-linearity in the elimination of thioridazine at high dose.

Multiple-dose pharmacokinetics of clozapine in patients.

After a 2-day buildup, patients were dosed continuously with clozapine solution at three ascending dose levels (37.5, 75, and 150 mg bid for 7 days at each dose level). Following the morning administration on the twenty-third day of dosing a drug holiday was instituted which lasted for a minimum of 48 hr. Serial plasma samples were obtained during each of the periods and during the drug holiday for the calculation of the steady-state parameters AUCSS, CSSmax, and CSSmin at each dose level as well as for the assessment of the terminal elimination rate. Mean parameter values for AUCSS, CSSmax, and CSSmin showed a linearly increasing response with the dose, well described by a straight line passing through the origin. The terminal elimination appeared to follow linear kinetics and had a mean half-life of 15.8 hr (range, 5.8-33 hr).

Respiratory proteins from the extremely thermophilic aerobic bacterium, Thermus thermophilus. Purification procedures for cytochromes c552, c555,549, and c1aa3 and chemical evidence for a single subunit cytochrome aa3.

We have developed a chemically defined, minimal growth medium for Thermus thermophilus which is suitable for nutritional studies, isotopic enrichment, and genetic manipulation of the organism. Reliable procedures are described for the large scale purification of cytochrome c552 from the periplasm and for cytochrome c555,549 and cytochrome c1 aa3 from the plasma membrane. In contrast to a previous report (Fee, J. A., Choc, M. G., Findling, K. L., Lorence, R., and Yoshida, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 147-151) which suggested a molecular weight near 200,000, the cytochrome c1aa3 complex was shown by protein and amino acid analyses to have Mr approximately 93,000. Sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography, combined with amino acid analyses, revealed the presence of only two proteins in a 1:1 ratio: C-protein has Mr approximately 33,000, binds heme C, and is thought to correspond to cytochrome c1. A-protein has Mr approximately 55,000 and is thought to bind the four redox components (2 heme A and 2 Cu) of cytochrome aa3.