Tacrolimus metabolite cross-reactivity in different tacrolimus assays

Objectives: Tacrolimus (FK506) is an immunosuppressive drug with great clinical promise. There is a controversy regarding the role of tacrolimus metabolites in immunosuppression and toxicity, and immunoassays and immunophilin binding assays have not been adequately tested for metabolite cross-reactivity. Methods are limited to HPLC and HPLC-MS for quantifying the parent drug. Mixed lymphocyte culture assay (MLC) is the preferred functional bioassay for the measurement of parent drug and active metabolites but it is not practical for routine laboratory use. Due to differences in assay methods and reagent specificity, the concentration of tacrolimus in a given specimen may vary among different assay kit manufacturers. The objective of this study was to evaluate the degree of cross-reactivity or interference of the three first-generation tacrolimus metabolites [13-O-demethyl (M-I), 31-O-demethyl (M-II) and 15-O-demethyl (M-III)] among two different tacrolimus immunoassays (Immunoassay: PRO-Trac II FK506, Abbott IMx tacrolimus-II); and the radioreceptor assays (RRA) using minor immunophilins (14, 37, and 52 kDa immunophilins) and tacrolimus binding protein (FKBP12).

Methods: First-generation tacrolimus metabolites (M-I, M-II, and M-III) spiked in drug-free whole blood were assayed with RRA using three minor immunophilins (14, 37, and 52 kDa) and two commercial immunoassay procedures (Incstar PRO-Trac II tacrolimus, Abbott IMx tacrolimus II). The results were compared to previously published FKBP-12 RRA data and their immunosuppressive potency.

Results and conclusion: The first generation tacrolimus metabolites (M-I, M-II, and M-III) were tested using concentrations of 10 and 20 ng/mL. The significance of the metabolite interference (% of the total interference) was calculated based on the relative concentration of each metabolite present at steady-state trough concentrations in renal transplant recipients [22]. Metabolite I, which has no functional immunosuppressive activity showed minimal interference compared to M-II and M-III in all assays except the 14 kDa RRA. The Incstar PRO-Trac II tacrolimus assay showed the least M-I interference. Metabolite-II, which has a pharmacologic potency similar to the parent drug, showed a significant interference in the immunoassays and significant interference in radioreceptor assays. Metabolite III, which is pharmacologically inactive, produces 3–10% interference in the different assays if its presence in the blood is 6% of the parent drug. The total interference from these three metabolites was greater in the immunoassays than in the receptor assays. Receptor assays for tacrolimus provide results closer to the target value than do immunoassays.     

Acute vascular rejection is associated with up-regulation of vitronectin receptor (alphavbeta3), increased expression of tissue factor, and activation of the extracellular matrix metalloproteinase induction system.

BACKGROUND: A cascade of inflammatory reactions characterize acute vascular rejection after heart transplantation. This study was undertaken to test the hypothesis that acute vascular rejection is associated with up-regulation of vitronectin receptor (alphavbeta3), increased expression of tissue factor, and activation of the extracellular matrix metalloproteinase induction system. METHODS: Acute vascular rejection developed in 14 heart transplant recipients within 2 weeks of transplantation, confirmed by immunofluorescence (AVR group). We compared these patients with 10 transplant recipients who had no evidence of acute vascular rejection or peritransplant ischemic injury (control group). We evaluated endomyocardial biopsy specimens for alphavbeta3, tissue factor, and extracellular matrix metalloproteinase inducer (EMMPRIN). RESULTS: Compared with the control group, the AVR group demonstrated evidence of significantly increased expression of alphavbeta3 (1.9-fold, p < 0.001), tissue factor (1.8-fold, p < 0.001), and EMMPRIN (1.5-fold, p < 0.001). All patients in the AVR group received plasmapheresis; 11 of 14 patients had evidence of ischemic necrosis on biopsy specimens, and 3 of 14 patients experienced hemodynamic compromise and graft dysfunction and died within 3 weeks of transplant. Another patient died at 10 months after transplant. CONCLUSIONS: Acute vascular rejection is associated with up-regulation of alphavbeta3, tissue factor, and activation of the matrix metalloproteinase induction system, which may contribute to the lethal morbidity associated with this disease.     

Localization of tissue plasminogen activator mRNA in adult rat brain

The distribution of tissue plasminogen activator (tPA) messenger RNA in rat brain was studied using in situ hybridization with ³⁵S UTP-labeled RNA probes derived from a fulllength tPA cDNA. Sense strand controls produced low, even backgrounds, with small elevations in the hippocampus. Full-length antisense probes produced strong signals over cerebral ventricular ependyma (including ependyma of the subcommissural organ), meninges, blood vessels, and Purkinje cell layer of the cerebellum, as well as strong signals over scattered cells throughout the brain. Some of these scattered labeled cells were large with lightly stained nuclei, while others were small with darkly stained nuclei. The large labeled cells, which were probably neurons, constituted 8% and 8% of cells in the brain stem and neocortex, respectively, and 100% of Purkinje cells. The small cells, which were present in all areas of the brain, constituted 3–11 % of cells in individual brain areas.     

'Diaphragmlike' stricture and ulcer of the colon during diclofenac treatment.

Diclofenac sodium is a widely used enteric-coated nonsteroidal anti-inflammatory drug. We describe a woman with Hemoccult-positive stools and iron deficiency anemia who developed both a colonic ulcer and a "diaphragm-like" colonic stricture while taking enteric-coated diclofenac. These lesions were evident on colonoscopy but not on barium studies. Biopsy specimens of the ulcer and stricture revealed particulate matter that was indistinguishable from diclofenac pill fragments by electron microscopy. Discontinuation of diclofenac therapy resulted in resolution of anemia and Hemoccult-positive stools. We conclude that (1) enteric-coated diclofenac is associated with both colonic ulcers and diaphragm-like colonic strictures; (2) the pathophysiologic mechanism for the development of both ulcers and strictures may involve a direct action of diclofenac within these lesions; (3) colonoscopy may be superior to barium studies in evaluating patients receiving diclofenac who have iron deficiency anemia and/or Hemoccult-positive stools.     

Anti-conflict efficacy of buspirone following acute versus chronic treatment

In many animal studies, acute treatment with the novel anxiolytic agent buspirone exhibits only minimal anxiolytic efficacy (i.e., increases in punished responding) when compared to benzodiazepines and barbiturates. The present studies examined the effects of acute pre-test challenges with buspirone in subjects receiving chronic post-test buspirone or saline treatments. Chronic post-test treatment with buspirone (4 mg/kg/day for 4 weeks, followed by 8 mg/kg/day for 12 weeks) did not significantly affect CSD behavior. Consistent with previous reports, acute pre-test administration of buspirone (0.125–2 mg/kg, IP) to subjects receiving chronic post-test saline treatment resulted in only a modest anti-conflict effect in the CSD paradigm (approximately ten shocks over control). In contrast, subjects chronically treated with buspirone exhibited a dramatically greater anti-conflict effect following acute challenge with buspirone (up to 40 shocks over control). These data are consistent with the hypothesis that the full anxiolytic efficacy of buspirone requires repeated administration.