Valerian C. Dias

Hyperhomocysteinemia and inflammatory bowel disease: prevalence and predictors in a cross-sectional study

OBJECTIVE:Homocysteine is a sulfur-containing amino acid formed during the demethylation of methionine. Vitamin B12 and folate deficiency and therapy with antifolate drugs may predispose patients with inflammatory bowel disease (IBD) to hyperhomocysteinemia. The known associations between hyperhomocysteinemia and smoking, osteoporosis, and thrombosis make it an interesting candidate as a pathogenetic link in IBD. The aim of this study was to identify the prevalence and risk factors of hyperhomocysteinemia in patients with IBD.METHODS:Sixty-five consecutive IBD patients were recruited from a tertiary outpatient gastroenterology practice. Fasting plasma homocysteine levels were measured, along with vitamin B12 and folate. Data regarding medication use, multivitamin use, disease location and severity, and extraintestinal manifestations of IBD were gathered. Homocysteine levels in 138 healthy control subjects were compared with the IBD cohort, and adjustments for age and sex were made using logistic regression. Multivariate analysis was performed to seek predictors of homocysteine levels.RESULTS:The mean age in the IBD cohort was 42 ± 13.4 yr (±SD), and 43% were male. The mean disease duration was 13.8 ± 9.4 yr, and 32% had used steroids within the last 3 months. Immunomodulator therapy had been used in 32%, and 75% had had an intestinal resection. Osteoporosis was present in 33% of patients. Five patients had experienced venous thrombosis or stroke, but only one of these had hyperhomocysteinemia. Of the 10 IBD patients (15.4%) with hyperhomocysteinemia, only two had vitamin B12 deficiency. The homocysteine levels in the IBD cohort cases and controls were 8.7 and 6.6 μmol/L, respectively (p < 0.05). IBD significantly increased the risk of hyperhomocysteinemia (adjusted odds ratio = 5.9 [95% CI: 1.5–24]). Advanced age, male sex, vitamin B12 deficiency or lower vitamin B12 serum levels, and multivitamin therapy were independently associated with higher homocysteine levels in the multivariate analysis (R2= 0.55; p = 0.001).CONCLUSIONS:Hyperhomocysteinemia is significantly more common in patients with IBD compared with healthy controls, and is associated with lower (but not necessarily deficient) vitamin B12 levels.

Blood distribution and single-dose pharmacokinetics of leflunomide.

Summary Leflunomide (HWA 486, LEF) is a novel isoxazole derivative with potent immunosuppressive properties. LEF is converted to its active metabolite (A77 1726) after absorption. Presently, the blood distribution and pharmacokinetics of LEF have not been reported. Such information would prove invaluable in determining the appropriate medium for analysis and optimal immunosuppressive dosing regimes. In this study, A77 1726 was found to be primarily associated (>95%) with the lipoprotein free fraction of plasma at all tested concentrations ranging from 0.4 to 100 mg/L. Detectable levels of A77 1726 (0.34 ± 0.18 mg/L), analyzed by HPLC, were found in the plasma free fraction only at the highest tested concentration (100 mg/L). Single-dose phar-macokinetics of A77 1726 (i.v.) and HWA 486 (p.o.) were investigated in five healthy New Zealand white rabbits. The half-lives (t1/2) of A77 1726 i.v. and HWA 486 p.o. administration were 3.88 ± 2.3 and 3.18 ± 1.6 h, respectively. The volume of distribution by both routes of administration indicates minimal distribution into tissues (Vdssp.o. = 0.14 ± 0.03 L/kg and Vdssi.v. = 0.09 ± 0.02 L/kg). The mean residence time of A77 1726 was greater after oral administration of LEF (MRTp.o. = 10.54 ± 2.6 h and MRTi.v. = 6.76 ± 1.0 h). Identical areas under the curve suggest bioavailability was 100% (AUCp.o. = 421.16 ± 204.5 mg ± h/L and AUCi.v. = 399.75 ± 126.9 mg ± h/L).

Measurement of the active leflunomide metabolite (A77 1726) by reverse-phase high-performance liquid chromatography

The immunosuppressive activity of leflunomide is expressed after conversion to its pharmacologically active metabolite A77 1726. Leflunomide is a potent immunosuppressant that inhibits both T-cell and B-cell activity. To date, no pharmacokinetic data have been reported on leflunomide or A77 1726, primarily because of lack of a suitable method for its analysis. We describe here the development and evaluation of a reverse-phase high-performance liquid chromatographic (HPLC) method for the analysis of A77 1726 in whole blood or plasma from humans or rabbits. In human blood, the method exhibited good analytic recoveries from 78 ± 13.5% to 108 ± 4.8% (mean ± SD) for drug concentrations ranging from 400 to 100,000 μg/L. When using a sample volume of 0.25 ml the sensitivity of the method was found to be 400 μg/L, with a working standard range of up to 200,000 μg/L. The sensitivity of the method can be increased to 40 μg/L when 1.0 ml of sample is used. Between-run coefficients of variation of 12.2 and 14.7% at A77 1726 mean concentrations of 1,006 and 8,146 μg/L were found for this method. No significant differences in recovery of drug were noted when either human or rabbit plasma or whole blood was used as the medium of analysis. In whole-blood specimens, A77 1726 was found to be stable for up to 10 days at −20 or −70°C.

Investigation of rapamycin transport and uptake across absorptive human intestinal cell monolayers

An in vitro intestinal cell culture model was used to characterize and investigate factors affecting uptake and transport of rapamycin (RAPA), a potent immunosuppressive drug. Studies were performed on three human intestinal cell monolayers (Caco-2, HCT-8, and T84), grown on microporous membrane inserts for 12 days. RAPA transport in all three monolayers was found to be dose dependent. The highest rates of transport were found at the highest tested final RAPA concentration of 10,000 micrograms/L. Apical to basal RAPA transport was linear in Caco-2 cell monolayers for up to 60 min, and in HCT-8 and T84 cell monolayers for up to 120 min. Temperature sensitive RAPA transport was found because incubation at 4 degrees C markedly attenuated transport by 97, 90, and 78% for Caco-2, HCT-8, and T84 monolayers, respectively. In all three monolayers RAPA transport was highly polarized because the apical to basal transport was greater than that in the opposite direction. RAPA uptake and transport across cell monolayers were compared when 10,000 micrograms/L of RAPA (cold) plus 0.05 microCi 14C-RAPA was added in combination with varying final concentrations (1,000, 10,000, and 100,000 micrograms/L) of the immunosuppressive drugs, CsA or RS. Increasing concentrations of CsA resulted in a significant dose-dependent decrease in 14C-RAPA transport across cell monolayers. In contrast, at high (100,000 micrograms/L) RS concentrations, 14C-RAPA transport was significantly increased. Uptake of 14C-RAPA into cell monolayers was significantly decreased only with the 100,000 micrograms/L CsA concentration. These studies suggest that combinations of immunosuppressive drugs given orally have a potential for altering the intestinal transport and uptake of RAPA.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of paraproteins and rheumatoid factor on four commercial immunoassays for vancomycin: implications for laboratorians and other health care professionals.

Background: Paraproteins, immunoglobulins (Igs), which are elevated in various autoimmune disorders, are known to interfere with various laboratory immunoassays, including vancomycin (VANC). Rheumatoid factor (RF), a known immunoassay interferant, may cause falsely elevated results. Objectives: The aims of this study were to (1) evaluate the effect of 3 paraproteins (IgA, IgG, and IgM) on 4 commercial VANC immunoassays [fluorescence polarization immunoassay; enzyme multiplied immunoassay; 2 particle-enhanced turbidimetric inhibition immunoassays]; (2) determine the concentration at which the effect is obtained, and (3) examine the influence of RF on the VANC methods. Method: Serum and plasma pools from patients prescribed VANC and a spiked VANC pool (20 mg/L) were each mixed 1:1 with individual patient specimens containing IgA (6–63 g/L), IgG (6–54 g/L), IgM (3–30 g/L) (n = 4 for each Ig), and a patient RF pool (196 IU/L). The mixtures (n = 39) were split and distributed for VANC analysis. Results: IgA and IgG in serum and plasma did not affect any of the VANC immunoassays. RF added to plasma specimens did not interfere, but in serum, elevated VAN results were observed. IgM did not affect the fluorescence polarization immunoassay and enzyme multiplied immunoassay methods but did attenuate VANC concentrations by both particle-enhanced turbidimetric inhibition immunoassays (Siemens, Beckman Coulter), with a more pronounced effect on the latter, producing concentrations >20% lower than expected in the patient serum and spiked plasma pools. The effect was progressively negative at effective IgM concentrations of 10 and 15 mg/L. Conclusions: This phenomenon is a major analytical and clinical issue that must be communicated to health care professionals caring for patients receiving VANC, so optimal therapy is achieved.

Evaluation of the CLINITEK ATLAS for routine macroscopic urinalysis.

OBJECTIVESnTo evaluate the performance of a new, benchtop, fully automated urine analyzer the CLINITEK ATLAS and compare it with the URICHEM 1000 CHEMSTRIP UA analyzer. Macroscopic analysis included measurement of 8 urine analyte chemistries and specific gravity by the refractive index method (SgRl).nnnMETHODSnThe analytical performance studies conducted were calibration stability, precision (within-run and day-to-day), comparison of results of 437 fresh patient urine specimens, analysis of time performance, and problem logging over a 16-day evaluation period.nnnRESULTSnSatisfactory calibration reproducibility, within-run (n = 10), and day-to-day (n = 16) precision was found because results fell within the +/- one color-block by the proposed National Committee for Clinical Laboratory Standards (NCCLS) criteria. Patient results (n = 437) from the 2 analyzers giving the same color-block agreement was found to be for pH, 52%; glucose, 92%; ketones, 86%; protein, 79%; bilirubin, 97%; leukocytes, 72%; blood, 80%; and nitrite, 98%. The concordance defined by the NCCLS criteria as the agreement of results +/- one color-block between the 2 analyzers was found to be for pH, 96%; glucose, 99%; ketones, 100%; protein, 95%; bilirubin, 100%; leukocytes, 97%; and blood 86%. The SgRl determined on ATLAS was correlated with the RD-10 Rapid Density analyzer with the following results: slope = 0.97, intercept = 0.033, r = 0.94, Syx = 0.003, for a range of values from 1.002 to 1.070.nnnCONCLUSIONnOur preliminary data indicate that the analytical performance, and automatable features for complete walk-away function of this analyzer can significantly increase the overall testing efficiency in the urinalysis laboratory.

The EMIT Cyclosporine Assay: development of application protocols for the Boehringer Mannheim Hitachi 911 and 917 analyzers.

OBJECTIVEnThe purpose of this work was to develop applications for the EMIT Cyclosporine (CsA) Assay on the Hitachi 911 and 917 analyzers.nnnMETHODS AND RESULTSnInstrument settings were optimized to arrive at the following assay characteristics on the Hitachi 917. Limit of sensitivity was 50 micrograms/L. Intra-assay coefficients of variation (CV) were 8.1% (n = 20; mean = 62 micrograms/L) and 4.2% (n = 20; mean = 315 micrograms/L), while interassay CVs were 13.0% (n = mean = 73 micrograms/L) and 5.7% (n = 43; mean = 391 micrograms/L). Recoveries of 95-104% were obtained by spiking aliquots of 3 whole blood patient pools of known CsA concentrations with CsA. Serial dilutions of 3 patient specimens demonstrated linear relationships between expected and actual CsA concentrations (r = 0.99, 0.99, 0.98; regression lines: y = 1.19x -17.1; y = 0.75x + 18.0; y = 1.01x + 3.7). Specimen carryover was not evident. Calibration stability is at least 10 days. Comparable assay characteristics were found for the Hitachi 911. Sequentially-collected trough whole blood specimens from renal (n = 3), liver (n = 3) and heart (n = 4) transplant patients prescribed CsA were collected up to 78 days post-transplant and analyzed by EMIT on the Hitachi 917 and also by fluorescence polarization immunoassay (FPIA) and high performance liquid chromatography (HPLC). The following linear regression equations were produced for the renal [EMIT = 0.801 (TDx) + 4.98, r = 0.91, Sy/x = 32, n = 37; EMIT = 0.877 (HPLC) + 56, r = 0.87, Sy/x = 38, n = 37]; liver [EMIT = 0.808 (TDx) - 27, r = 0.94, Sy/x = 42, n = 37; EMIT = 0.953 (HPLC) + 44, r = 0.89, Sy/x = 57, n = 37] and heart [EMIT = 0.820 (TDx) - 24, r = 0.94, Sy/x = 31, n = 45, EMIT = 0.956 (HPLC) + 54, r = 0.91, Sy/x = 38, n = 45] patient samples. FPIA values average 32% more than EMIT-derived CsA concentrations on the Hitachi 917, which in turn averaged 15% more than HPLC values. In addition, these levels were compared intra-individually. CsA concentrations within all patients were significantly higher (p < 0.05, paired t-test) by FPIA compared to EMIT and by FPIA compared to HPLC. Although CsA concentrations within most patients were significantly higher (p < 0.05) by EMIT compared to HPLC, levels determined in 4 transplant patients (1 renal, 1 liver, 2 heart) were not different.nnnCONCLUSIONnDevelopment of applications for the EMIT CsA Assay on two highly automated, random access instruments, the Hitachi 911 and Hitachi 917, enhances the versatility of the immunoassay for routine therapeutic drug monitoring of this immunosuppressant in the clinical setting.

Orally administered immunosuppressants modify intestinal uptake of nutrients in rabbits

The effect on intestinal nutrient transport of the immunosuppressive drugs cyclosporin A (CsA), cyclosporin G (CsG), and rapamycin (RAP) was determined in New Zealand white rabbits. Rabbits received oral doses of CsA (20 mg/kg/day), CsG (10 mg/kg/day), or RAP (1 mg/kg/day) for 10 days. Animals receiving RAP had decreased food intake and weight gain compared with controls. This correlated with a decrease in both total ileal weight and corresponding mucosal weight. CsA and CsG administration had no effect on food intake, total weight gain, or intestinal weight. Villus surface area was significantly decreased in all groups as compared with controls. Jejunal uptake of D-glucose as well as 1 medium and 4 long chain fatty acids was not affected by drug administration, while both mucosal-to-serosal and net 3–0-methylglucose fluxes were increased (P<0.05) in the jejunum by all 3 drugs. In the ileum, the rates of uptake of D-glucose as well as stearic and linoleic acids were increased in animals treated with RAP compared with controls. There was an increase in the ileal values of the maximal transport rate (Vmax) and apparent Michaelis constant (Km*) in RAP, and a fall in the Vmax and Km* in CsG. CsG administration resulted in a decreased cholesterol uptake in both jejunum and ileum, and a decreased D-glucose uptake in the ileum compared with controls. These differences in glucose uptake among groups could not be attributed to variations in body, intestinal, or mucosal weights. It is unlikely that the changes observed in CsA- and CsG-treated animals would have nutritional importance, as these animals gained weight normally. In addition, in these animals the changes mainly occurred in the ileum, not in the jejunum, where most glucose is absorbed, and the associated alterations in the values of the Vmax and Km* would lead to reciprocal changes in the rates of uptake of varying luminal concentrations of glucose. In contrast, these changes are likely to be of more importance in RAP-treated animals, since they failed to gain weight normally. The significance of these findings needs to be established in chronically treated animals.

An in vitro method for predicting in vivo oral bioavailability of novel immunosuppressive drugs.

OBJECTIVEnTo evaluate an in vitro method for predicting oral availability of novel immunosuppressive drugs, cyclosporine A (CsA) and rapamycin (RAPA).nnnMETHODSnIn this study, we report the development and characterization of an in vitro method to study the influence of vehicle composition on cyclosporine A (CsA) and rapamycin (RAPA) drug efflux across 12 days postconfluent, absorptive human Caco-2 intestinal epithelial cell monolayers. The apical-to-basal (Jab) and the basal-to-apical (Jba) fluxes of 0.5 muCi 3H-CsA or 0.05 muCi 14C-RAPA solubilized in a 10 mg/L final drug concentration in vehicle were measured.nnnRESULTSnThe Jab CsA flux was found to be dose dependent, temperature sensitive, and highly polarized (Jab > Jba). For CsA the vehicles were Neoral, Sandimmune, 95% (v/v) ethanol/fetal bovine serum (ethanol/FBS); and for RAPA these were polyethylene glycol/dimethylacetamide (PEG/DMA), polysorbate/Phosal PEG, ethanol/FBS. When Neoral-CsA was tested, the Jab flux of 3H-CsA was the highest and increased almost linearly even after an incubate time of 240 min. The Jab flux of 3H-CsA when Sandimmune-CsA or ethanol/FBS-CsA were used as vehicle was lower and reached a maximal rate by 120 min. In contrast the Jab flux of 14C-RAPA using either PEG/DMA-RAPA or ethanol/FBS-RAPA as vehicle was highest and reached a maximal rate by 120 min, in contrast to the polysorbate/Phosal PEG-RAPA vehicle, which was significantly lower.nnnCONCLUSIONnThese data are consistent with the pharmacokinetics of these ISD reported in vivo in human patients or in rabbits, using the same vehicles in the oral formulation. As an integral part of drug development, the data presented that an in vitro system as described may be useful in predicting the effect of drug vehicle on absorption in vivo.