A new highly specific buprenorphine immunoassay for monitoring buprenorphine compliance and abuse.

Urine buprenorphine screening is utilized to assess buprenorphine compliance and to detect illicit use. Robust screening assays should be specific for buprenorphine without cross-reactivity with other opioids, which are frequently present in patients treated for opioid addiction and chronic pain. We evaluated the new Lin-Zhi urine buprenorphine enzyme immunoassay (EIA) as a potentially more specific alternative to the Microgenics cloned enzyme donor immunoassay (CEDIA) by using 149 urines originating from patients treated for chronic pain and opioid addiction. The EIA methodology offered specific detection of buprenorphine use (100%) (106/106) and provided superior overall agreement with liquid chromatography-tandem mass spectrometry, 95% (142/149) and 91% (135/149) using 5 ng/mL (EIA[5]) and 10 ng/mL (EIA[10]) cutoffs, respectively, compared to CEDIA, 79% (117/149). CEDIA generated 27 false positives, most of which were observed in patients positive for other opioids, providing an overall specificity of 75% (79/106). CEDIA also demonstrated interference from structurally unrelated drugs, chloroquine and hydroxychloroquine. CEDIA and EIA[5] yielded similar sensitivities, both detecting 96% (22/23) of positive samples from patients prescribed buprenorphine, and 88% (38/43) and 81% (35/43), respectively, of all positive samples (illicit and prescribed users). The EIA methodology provides highly specific and sensitive detection of buprenorphine use, without the potential for opioid cross-reactivity.

Effect of tramadol use on three point-of-care and one instrument-based immunoassays for urine buprenorphine.

We report that use of the popular analgesic tramadol can cause false-positive urine buprenorphine results. We examined the extent of tramadol cross-reactivity in three point-of-care urine buprenorphine immunoassays (ACON, QuikStrip, and ABMC) and an instrument-based one (Cedia). We tested 29 urine samples from patients known to be taking tramadol. Ten different samples tested positive for urine buprenorphine by at least one immunoassay. Samples with positive buprenorphine screens by immunoassay were tested for total buprenorphine and total norbuprenorphine content by liquid chromatography-tandem mass spectrometry (LC-MS-MS), which confirmed that seven of the 10 positive samples were false-positives. The remaining three positive immunoassay samples had insufficient quantity for LC-MS-MS testing. No false-positives were detected with the ACON (10 ng/mL calibration cutoff) or the Cedia assay (using a 20 ng/mL calibration cutoff). All four false-positive Cedia results (using a 5 ng/mL cutoff) in this study tested negative using the ACON device. Our data suggest that tramadol use can cause false-positive urine buprenorphine immunoassays, and this effect appears to be assay-dependent. Tramadol interference with the Cedia assay is clinically relevant, especially if the 5 ng/mL calibration cutoff is used.

Buprenorphine ameliorates the effect of surgery on hypothalamus-pituitary-adrenal axis, natural killer cell activity and metastatic colonization in rats in comparison with morphine or fentanyl treatment.

Not all opioids employed in clinical practice share the same immunosuppressive properties. The potent partial micro-agonist buprenorphine appears to exhibit a neutral effect on the immune responses. Surgery stress is associated with decreased natural killer cell activity (NK) and enhancement of tumor metastasis in rats. We analyzed the ability of buprenorphine to prevent the effects of experimental surgery on HPA activation (plasma corticosterone levels), NK activity and lung diffusion of the NK sensitive tumor MADB106. Buprenorphine (0.1mg/kg) was compared with equianalgesic doses of fentanyl (0.1mg/kg) and morphine (10mg/kg) in this animal model. In normal animals morphine and fentanyl stimulate the HPA axis, decrease NK activity and augment tumor metastasis, while buprenorphine is devoid of these effects. Surgery significantly raised corticosterone levels, suppressed NK activity and increased MADB106 metastasis. Only buprenorphine was able to prevent the neuroendocrine and immune system alterations and ameliorate the increase of tumor metastasis induced by surgical stress. These preclinical findings suggest that an adequate treatment of surgically induced stress immunosuppression with an opioid drug devoid of immunosuppressive effects may also play a protective role against the metastatic diffusion following cancer surgery.

Evaluation of buprenorphine CEDIA assay versus GC-MS and ELISA using urine samples from patients in substitution treatment.

As buprenorphine becomes more clinically used in heroin substitution treatment, there is an increasing need for methods suitable for high-volume screening. In this study, a new immunochemical test based on CEDIA technology was evaluated for the use in clinical urine drug testing. The method was compared with an existing ELISA method and a gas chromatography-mass spectrometry (GC-MS) method on urine specimens from patients in heroin substitution treatment. The precision of the CEDIA assay was < 9% both within- and between-day at levels at and above the cutoff limit of 5 microg/L. The concordance in qualitative results with an existing ELISA method was 96.8%. The CEDIA measuring range was extended by diluting urine samples 100-fold with saline, and the results agreed well (slope of regression line was 1.09, r(2) = 0.968) with GC-MS. The sensitivity of CEDIA in detecting authentic specimen containing buprenorphine at levels >or= 5 microg/L was 99.5%. Cross-reactivity causing false-positive response was discovered in patients receiving prescribed dihydrocodeine. The urine concentration of total buprenorphine in urine from patients prescribed daily doses between 0.2 and 24 mg ranged from 0.5 to 2900 microg/L. The concentration of the metabolite norbuprenorphine was usually higher, and the median ratio of buprenorphine to norbuprenorphine was 0.23 (95% were below 1). We conclude that the CEDIA assay is suitable for application in high-volume screening of buprenorphine for urine drug testing.

Cross-reactivity of the CEDIA buprenorphine assay with opiates: an Austrian phenomenon?

When testing the Microgenics CEDIA assay for immunological buprenorphine analysis, cross-reactivity between the buprenorphine reagents and opiates was observed at concentrations higher than 120 mg/l morphine, 320 mg/l methadone, 30 mg/l codeine, 60 mg/l dihydrocodeine and 520 mg/l morphine-3-glucuronide. The cross-reactivity with morphine has the greatest impact on routine screening as opiate maintenance therapy in Austria is also performed with slow-release oral morphine. The use of a second cutoff value of 30 mug/l for urine samples that are (immunologically) positive for opiates is therefore suggested, compared to the cutoff value of 5 microg/l proposed by the manufacturer.

No detrimental effect from chronic exposure to buprenorphine on corticosteroid-binding globulin and corticosensitive immune parameters.

Opioid drugs reportedly regulate the immune system via their effects on the hypothalamic- pituitary-adrenal (HPA) axis. The present study was carried out to assess the effects of chronic exposure to buprenorphine on HPA axis activation, corticosteroid-binding globulin (CBG), the main glucocorticoid (GC) carrier, and the immune system. Results show that buprenorphine, delivered by osmotic pump subcutaneously in C57BL/6 male mice during a 10-day period, caused a marked decrease in total corticosterone (CORT) levels at day 1 of exposure. CORT levels then increased with maximal values observed at day 5 of exposure. After day 5, total CORT levels gradually decreased and returned to control values. No significant changes were observed in CBG protein levels and mRNA expression in the liver. Since CBG levels remained unchanged, the percentage of free CORT values in buprenorphine mice did not differ from control values. Thus, the variations observed in the amount of free CORT were related only to changes measured in total CORT. These endocrine changes did not have a significant impact on the immune parameters measured. Total CD(4)+ and CD(8)+ splenic and thymic populations were not modulated by buprenorphine. However, splenocytes from mice exposed to buprenorphine after 5 days exhibited greater proliferation upon anti-TCR monoclonal antibody stimulation than saline-exposed mice. These results indicate that buprenorphine can be safely used because it did not have significant effects on GC availability for immune corticosensitive cells.

Development of a radioimmunoassay for the determination of buprenorphine in biological samples.

The development of a specific and sensitive radioimmunoassay for the detection of buprenorphine in urine samples is described. With minor adjustments, the assay was also applied to the analysis for buprenorphine in plasma samples. The 2-diazobenzoic acid derivative of buprenorphine has been prepared as a hapten. The immunization of rabbits with the hapten-bovine serum albumin conjugate resulted in the production of antibodies, which cross-reacted with N-dealkylbuprenorphine up to about the 90% level. The antibodies showed very low cross-reactivities with the 3-O-glucuronides and with the structural analogue etorphine. The assay was mainly used to pre-screen for buprenorphine in urine samples of persons suspected of Temgesic misuse and to determine buprenorphine in plasma samples. A linear calibration graph for buprenorphine was obtained after logit-log regression [Y = 0.383 (s, 0.059) - 0.535 X (s, 0.025); r = 0.997 (s, 0.001)]. The spiking recovery study showed a linear regression of Y (observed) = 0.94 + 0.84 X (expected); r = 0.997. Intra- and inter-assay relative standard deviations were < 4.35 and < 6.36%, respectively. A comparison study of the high-performance liquid chromatographic determination (X) to the radioimmunoassay (Y) resulted in the following regression equation for the urine samples: Y = 1.44 + 1.64 X (n = 32; r = 0.910) and Y = 0.007 + 1.58 X (n = 10; r = 0.930) for plasma specimens. The minimum detectable dose of the immunoassay was calculated to be 10 pg ml-1 (Student's t-distribution, p = 0.01, degrees of freedom = 8).

Radioimmunoassay of buprenorphine with iodine label: analysis of buprenorphine and metabolites in human plasma.

Quantitative analysis of potent opiate drugs in plasma by radioimmunoassay is potentially inaccurate because of the occurrence of cross-reacting metabolites. This paper describes the chemical synthesis of buprenorphine-3-O-glucuronide, a metabolite of buprenorphine, and an extraction procedure coupled with radioimmunoassay which allows the sensitive and specific measurement of buprenorphine using an iodinated buprenorphine derivative. The measurement of extracted and unextracted samples using two different antisera allowed investigation of the metabolism of buprenorphine. In four patients who had taken sublingual buprenorphine for at least one month, N-dealkyl buprenorphine was present in similar concentrations to those of buprenorphine, while buprenorphine-3-O-glucuronide was present in two to three times those concentrations.

The radioimmunoassay of buprenorphine.

Antisera to buprenorphine were obtained in rabbits immunised with 3-0-carboxymethylbuprenorphine and N-hemisuccinyl-norbuprenorphine conjugated to bovine serum albumin. Using the latter antiserum and tritium labelled buprenorphine a radioimmunoassay have good accuracy and precision was developed for concentrations as low as 50 picograms in 1 ml of plasma. The N-hemisuccinyl antiserum crossreacted with norbuprenorphine, and the 3-0-glucuronide conjugate with the 3-0-carboxymethyl antiserum. Cross-reactivity of both antisera to other pharmacologically related compounds was negligible. The assay was employed to determine plasma buprenorphine concentration following its parenteral administration to dog and man.