Separation of blood microsamples by exploiting sedimentation at the microscale.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients.

Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.

Oral flurbiprofen metabolic ratio assessment using a single-point dried blood spot.

We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.

Stereoselective block of hERG channel by (S)-methadone and QT interval prolongation in CYP2B6 slow metabolizers.

Methadone inhibits the cardiac potassium channel hERG and can cause a prolonged QT interval. Methadone is chiral but its therapeutic activity is mainly due to (R)-methadone. Whole-cell patch-clamp experiments using cells expressing hERG showed that (S)-methadone blocked the hERG current 3.5-fold more potently than (R)-methadone (IC50s (half-maximal inhibitory concentrations) at 37 degrees C: 2 and 7 microM). As CYP2B6 slow metabolizer (SM) status results in a reduced ability to metabolize (S)-methadone, electrocardiograms, CYP2B6 genotypes, and (R)- and (S)-methadone plasma concentrations were obtained for 179 patients receiving (R,S)-methadone. The mean heart-rate-corrected QT (QTc) was higher in CYP2B6 SMs (*6/*6 genotype; 439+/-25 ms; n=11) than in extensive metabolizers (non *6/*6; 421+/-25 ms; n=168; P=0.017). CYP2B6 SM status was associated with an increased risk of prolonged QTc (odds ratio=4.5, 95% confidence interval=1.2-17.7; P=0.03). This study reports the first genetic factor implicated in methadone metabolism that may increase the risk of cardiac arrhythmias and sudden death. This risk could be reduced by the administration of (R)-methadone.

Cytochrome P450 2D6 genotype and methadone steady-state concentrations.

A genetic polymorphism of cytochrome P450 2D6 has been described with the existence of poor (zero functional genes), extensive (one or two functional genes), and ultrarapid metabolizers (three or more functional genes). The authors measured the steady-state trough (R)- (i.e., the active enantiomer), (S)-, and (R,S)-methadone plasma levels in opiate-dependent patients receiving methadone maintenance treatment (MMT) and genotyped them for cytochrome P4502D6. The patients' medical records were reviewed to assess the outcome of the MMT with regard to the absence of illicit opiate consumption and to the absence of withdrawal complaints in ultrarapid and poor metabolizers. Of 256 patients included, 18 were found to be poor metabolizers, 228 to be extensive metabolizers, and 10 to be ultrarapid metabolizers. Significant differences were found between genotypes for (R)- (p = 0.024), (S)- (p = 0.033), and (R,S)-methadone (p = 0.026) concentrations to dose-to-weight ratios. For (R)-methadone, a significant difference was found between ultrarapid metabolizers and poor metabolizers (p = 0.009), with the median value in the former group being only 54% of the median value in the latter group. These results confirm the involvement of cytochrome P450 2D6 in methadone metabolism. Although the difference was nonsignificant (p = 0.103), 13 (72%) of the 18 poor metabolizers and only 4 (40%) of the 10 ultrarapid metabolizers were considered successful in their treatment. More studies are needed to examine the influence of the ultrarapid metabolizer status on the outcome of the MMT.

Double-blind randomized trial of buprenorphine and methadone in opiate dependence.

This study compared the safety and efficacy of sublingual buprenorphine tablets with oral methadone in a population of opioid-dependent individuals in a double-blind, randomized, 6-week trial using a flexible dosing procedure. Fifty-eight patients seeking treatment for opioid dependence were recruited in three outpatient facilities and randomly assigned to substitution with buprenorphine or methadone. The retention rate was significantly better in the methadone maintained group (90 vs. 56%; P<0.001). Subjects completing the study in both the treatment groups had similar proportions of opioid positive urine samples (buprenorphine 62%; methadone 59%) and positive urine specimens, as well as mean heroin craving scores decreased significantly over time (P=0.035 and P<0.001). The proportion of cocaine-positive toxicology results did not differ between groups. At week six mean stabilization doses were 10.5 mg per day for the sublingual buprenorphine tablet, and 69.8 mg per day for methadone, respectively. Patient performance during maintenance was similar in both the groups. The high attrition rate in the buprenorphine group during the induction phase might reflect inadequate induction doses. Thus, buprenorphine is a viable alternative for methadone in short-term maintenance treatment for heroin dependence if treatment induction is done with adequate dosages.

Plasma concentrations of the enantiomers of methadone and therapeutic response in methadone maintenance treatment.

Methadone is a 50:50 mixture of two enantiomers and (R)-methadone accounts for the majority of its opioid effect. The aim of this study was to determine whether a blood concentration of (R)-methadone can be associated with therapeutic response in addict patients in methadone maintenance treatment. Trough plasma concentrations of (R)-, (S)- and (R,S)-methadone were measured in 180 patients in maintenance treatment. Therapeutic response was defined by the absence of illicit opiate or cocaine in urine samples collected during a 2-month period prior to blood sampling. A large interindividual variability of (R)-methadone concentration-to-dose-to-weight ratios was found (mean, S.D., median, range: 112, 54, 100, 19-316 ng x kg/ml x mg). With regard to the consumption of illicit opiate (but not of cocaine), a therapeutic response was associated with (R)- (at 250 ng/ml) and (R,S)-methadone (at 400 ng/ml) but not with (S)-methadone concentrations. A higher specificity was calculated for (R)- than for (R,S)-methadone, as the number of non-responders above this threshold divided by the total number of non-responders was higher for (R,S)-methadone (19%) than for (R)-methadone (7%). The results support the use of therapeutic drug monitoring of (R)-methadone in cases of continued intake of illicit opiates. Due to the variability of methadone concentration-to-dose-to-weight ratios, theoretical doses of racemic methadone could be as small as 55 mg/day and as large as 921 mg/day to produce a plasma (R)-methadone concentration of 250 ng/ml in a 70-kg patient. This demonstrates the importance of individualizing methadone treatment.

[Forensic medicine experiences with methadone substitution in the Geneva canton]. / Rechtsmedizinische Erfahrungen mit der Methadon-Substitution im Kanton Genf.

Methadone treatment for heroin addiction has followed three distinct periods in Geneva, Switzerland. The first period (1970-1979) corresponds to the beginning of the heroin addiction epidemic. Treatment was restricted to detoxification and did not succeed in reducing fatal overdoses. During the second period (1980-1989), methadone maintenance program was favoured but access to this program was limited. This period has brought a decrease of illegal heroin consumption and criminality but not of fatal overdoses. Finally, during the third period (since 1990), legislation was changed to allow easier access to methadone maintenance program. As a consequence there was a significant drop in lethal heroin overdoses and in deaths attributed to HIV.

Enantioselective analysis of methadone in saliva by liquid chromatography-mass spectrometry.

Saliva was tested and evaluated as a biological matrix for methadone (Mtd) monitoring. Conventional method using a narrow bore C18 column, and an enantioselective method using a narrow bore alpha1-acid glycoprotein column, were developed using liquid chromatography coupled with a mass spectromeric (MS) detector. After optimisation of MS conditions by flow injection analysis, selected ion monitoring detection was used to enhance sensitivity. The total Mtd concentration and the enantiomeric ratio in saliva were validated using an experimental design. The methods were applied to samples provided by heroin addicts undergoing a Mtd treatment. Results on total Mtd determination showed a very poor correlation between saliva and serum, whereas the enantiomeric ratios of Mtd gave a very good one.

Lethal methadone intoxications in Geneva, Switzerland, from 1994 to 1998.

AIMS: To estimate the number of methadone lethal intoxications in Geneva from 1994 to 1998, where the number of patients in methadone treatment has more than doubled since 1990. DESIGN: Retrospective study of all toxicological, autopsy and clinical data. SETTING: The Geneva Department of Forensic Medicine. PARTICIPANTS: All suspected overdose deaths in Geneva from 1994 to 1998. Cases were selected on the basis that the only cause of death was a potentially lethal drug concentration in the postmortem blood sample. MEASUREMENT: Toxicology and autopsy findings, clinical and drug history. FINDINGS: There were 106 lethal drug intoxications over the period. The overall number of drug intoxication deaths went from 33 in 1994 to nine in 1998. Thirty-six cases had methadone identified in their blood. All the 36 cases but one had medications or other drugs used illicitly present in the blood or urine. Of these 36 cases, 21 were attributed to methadone lethal intoxication. Only seven of these 21 decedents were enrolled in a methadone programmes. The number of deaths attributed to methadone intoxication ranged from three to five per year. CONCLUSION: Most lethal methadone intoxication is due to diverted or illegal methadone in association with medications or other drugs used illicitly. Furthermore, the increase in methadone prescription under strict medical control with health measures aimed at drug abuse prevention did not lead, in our study, to an increase of methadone lethal intoxication and may have been partly responsible for the large decrease of overall drug intoxication deaths during the time of our study.

Development of validated stereoselective methods for methadone determination in clinical samples.

A stereoselective analysis of methadone (Mtd) in whole blood and serum was developed using liquid chromatography on a protein based chiral stationary phase. Liquid-liquid extraction (LLE) and solid phase extraction methods were applied before chromatographic analysis. The extraction procedure, as well as the choice of the biological matrix, showed significant differences in the extraction yield and in the precision of the assays. Serum was selected for this assay and LLE was chosen as the preparation step because of its simplicity and rapidity. The total procedure was validated and applied to clinical samples. Samples taken from 45 heroin-addicted patients were analyzed. A correlation was found between the dose administered and Mtd concentration (total and R-form), but interindividual variability of the total normalized Mtd was seen (concentration varied from 90 to 530 ng/ml). Furthermore, two populations were apparently observed with a mean Mtd concentration of 200 and 475 ng/ml, respectively. Stereoselective analyses showed that more than 50% of the patients presented a nonracemic ratio, and particularly about 25% showed a preferential metabolism of the active R-Mtd enantiomer. Therefore, the stereoselective determination of Mtd is necessary to improve the quality of the treatment of heroin addiction.

Prevalence and incidence rate of HIV, hepatitis B and C among drug users on methadone maintenance treatment in Geneva between 1988 and 1995.

OBJECTIVES: To evaluate the prevalence and incidence rates of infection with HIV, hepatitis B (HBV) and hepatitis C (HCV), in a cohort of drug users (DU) in Geneva, Switzerland. DESIGN: Prospective open cohort study. SETTING: Private methadone maintenance treatment (MMT) programme. PATIENTS, PARTICIPANTS: Over 700 DU in treatment between 1988 and 1995 were tested biannually for HIV, HBV and HCV infection. INTERVENTION: None. MAIN OUTCOME MEASURE: Prevalence for HIV, HBV and HCV at study entry, determined by gender, by injection behaviour, by year of start of MMT and incidence rates for HIV, HBV and HCV, assuming equal risk of seroconversion on each day of the interval between last negative and first positive test. RESULTS: The prevalence at entry into treatment declined dramatically over time for all three viruses. Comparing DU entering treatment before 1988 to those entering treatment after 1993, the prevalence of HIV was 38.2% versus 4.5%, of HBV 80.5% versus 20.1%, and of HCV 91.6% versus 29.8%, respectively. Follow-up rate was 80%. The incidence rates for HIV and HBV were 0.6% and 2.1% per person-year of follow-up, respectively. For HCV the rate was higher (4.2%) especially among women (9.6%). CONCLUSION: These data suggest that DU have changed HIV risk-taking behaviour in response to HIV prevention campaigns. Current prevention efforts should focus on improvement of HCV prevention, identification of high-risk individuals and maintaining safe behaviour.

Comparison of buprenorphine and methadone in the treatment of opioid dependence. Swiss multicentre study.

A three-centre, randomised, double-blind study was designed to compare the efficacy and safety of buprenorphine and methadone. This was the first European study to compare these agents and was based on a previous trial performed in the US. Opioid-dependent subjects were randomised to receive either sublingual buprenorphine or oral methadone daily. Both objective and subjective measures of efficacy were monitored weekly, and safety parameters were regularly monitored over the entire six-week study. Urinalysis showed that the two treatments were similar with a slight increase in opioid-negative urines noted in both groups. The retention rate in the buprenorphine group was lower than in the methadone group, although it has been suggested that the buprenorphine dose may have been too low for some patients. None of the side effects noted were considered serious and all were attributable to chronic opioid dependence. Experience of two years substitution treatment in Fribourg suggests that initial induction onto buprenorphine allows for patients to be subgrouped before being given the most appropriate maintenance agent. Further investigation is required into the different dose-related effects of buprenorphine seen in particular subsets of addicts.

[Clinical aspects and evaluation of methadone substitution therapy]. / Clinique et évaluation des traitements de substitution à la methadone.

For a long time, regrettable prejudices have slowed down the expansion of methadone treatment. The arrival of AIDS and the need to find new therapies for addicts have facilitated a reduction in the misunderstanding concerning methadone. Methadone does not induce a narcotic, analgesic, or tranquilizing state for dependant addicts, is medically very safe and is not necessarily a life-long cure. Methadone treatments have become the most efficient means of treating addicts who are not able or willing to start short term abstinence programmes or go to specialized institutions. An evaluation of methadone treatments at the Phoenix Foundation in Geneva has confirmed that these programmes were able to reduce heroin use, delinquency, prostitution, risk of AIDS, overdoses or suicides and to maintain a good medical and psychosocial stability for the majority of the patients.

Absence of chronic human immunodeficiency virus infection without seroconversion in intravenous drug users: a prospective and retrospective study.

It has been reported that human immunodeficiency virus type 1 (HIV-1) infection may exist in persons without specific antibodies for years. To measure the frequency of a silent carrier state, a study was conducted in a cohort of 124 intravenous drug users (IVDUs) without anti-HIV-1 antibodies. All the participants had engaged in high-risk behavior for HIV-1 transmission for a number of years until 1987 or later. Samples were analyzed at 6-month intervals for the presence of HIV-1 provirus using DNA amplification and for the appearance of anti-HIV-1 antibodies. HIV-1 provirus and antibodies were undetectable in 122 participants, whereas seroconversion was observed in 2. In one of these, both amplified HIV-1 pol gene segment and anti-HIV-1 antibodies were detected simultaneously, and in the other, provirus was detected 1 month before seroconversion. This study suggests that long-term HIV-1 infection without anti-HIV-1 antibodies is rare and that repeated antibody testing is sufficient to determine the HIV-1 status of a person no longer at high risk for HIV-1 infection.

Loss of antibodies against hepatitis C virus in HIV-seropositive intravenous drug users.

A time-point survey of 262 ex-intravenous drug users (IVDUs) on methadone treatment showed serological evidence of hepatitis C virus (HCV) in 64%, hepatitis B virus in 74% and HIV in 36%. Analysis of previously collected frozen stored sera of the same patients showed that 31 further IVDUs had had anti-HCV antibodies in the past and had lost them in a time-dependent fashion. Most HCV seroreversion was detected in HIV-positive people: 20 out of 85 in the HIV-positive group versus 11 out of 18 in the HIV-negative group.