Drug-free Volunteers Research Articles

Simultaneous determination for 5 human urinary metabolites of UR-144 and XLR-11 by LC-MS/MS

This study aims to explore how the abused synthetic cannabinoids in human urine can be determined qualitatively and quantitatively by using the LC-MS/MS method. As a method validation based on the current FDA guideline, we tested 55 human urine samples (5 from drug-free volunteers and 50 from drug abusers) and evaluated the presence of parent drugs (UR144 and XLR11) and their five metabolites (XLR-11-N-4-Hydroxypentyl, XLR-11-6-Hydroxyindole, UR-144-N-4-Hydroxypentyl, UR-144-N-5-hydroxypentyl, and UR-144-N-pentanoic). The experimental data showed that the values of low limit of detection (LOD) for these analytes ranged from 0.05 to 0.15 ng/mL. Their low limit of quantitation (LLOQ) value was 1.0 ng/mL. The values of linear coefficients of determination (R2) for these analytes ranged from 0.9914 to 0.9988. Among these 50 urine testing samples of drug abusers, the parent XLR-11 and UR-144 analytes were detected only in 4 samples (with a concentration of 2.14 − 4.5 ng/mL) and 3 samples (with a concentration of 3.2 − 5.4 ng/mL), respectively. At the same time, the XLR-11-N-4-Hydroxypentyl (with a concentration of 4.5 − 57.2 ng/mL) and XLR-11-6-Hydroxyindole (with a concentration of 4.8 − 51.7 ng/mL) metabolites were detected in 24 and 11 samples, respectively. In addition, the UR-144-N-4-Hydroxypentyl (with a concentration of 3.5 − 87.24 ng/mL), UR-144-N-5-hydroxypentyl (with a concentration of 14.5-83.9 ng/mL), and UR-144-N-pentanoic (with a concentration of 2.5 − 104.4 ng/mL) metabolites were found in 26, 29, and 38 samples, respectively.

Noninvasive Detection of Cocaine and Heroin Use with Single Fingerprints: Determination of an Environmental Cutoff.

Recent publications have explored the possibility of using fingerprints to confirm drug use, but none has yet dealt with environmental contamination from fingertips. Here we explored the possibility of establishing an environmental cutoff for drug testing from a single fingerprint. Fingerprint samples (n = 100) were collected from the hands of 50 nondrug users before and after handwashing to establish separate environmental cutoff values and testing protocols for cocaine, benzoylecgonine, heroin, and 6-monoacetylmorphine. The cutoff was challenged by testing the fingerprints of drug-free volunteers after shaking hands with drug users. Fingerprints from patients who testified to taking cocaine (n = 32) and heroin (n = 24) were also collected and analyzed. A different cutoff value needed to be applied, depending on whether the fingerprints were collected as presented or after handwashing. Applying these cutoffs gave a 0% false-positive rate from the drug-free volunteers. After application of the cutoff, the detection rate (compared to patient testimony) for washed hands of patients was 87.5% for cocaine use and 100% for heroin use. Fingerprints show enhanced levels of cocaine, heroin, and their respective metabolites in patients who testified to taking the substances, compared with the population of naïve drug users surveyed, and a cutoff (decision level) can be established. The cutoff is robust enough to account for small increases in analyte observed after secondary transfer.

A Direct Immersion Solid-Phase Microextraction Gas Chromatography/Mass Spectrometry Method for the Simultaneous Detection of Levamisole and Minor Cocaine Congeners in Hair Samples From Chronic Abusers

Because levamisole has been increasingly found as a component of illicit drugs, a robust method to detect its presence in hair samples is needed. However, no systematic research on the detection of levamisole in hair samples has been published. The method presented here uses direct immersion solid-phase microextraction coupled with gas chromatography and mass spectrometry (DI-SPME-GC/MS) to detect levamisole and minor cocaine congeners in hair samples using a single-extraction method. Fifty hair samples taken in the last 4 years were obtained from cocaine abusers, along with controls taken from drug-free volunteers. Sampling was performed using direct immersion with a 30-μm polydimethylsiloxane fused silica/stainless steel fiber. Calibration curves were prepared by adding known amounts of analytes and deuterated internal standards to the hair samples taken from drug-free volunteers. This study focused on the adulterant levamisole and some minor cocaine congeners (tropococaine, norcocaine, and cocaethylene). Levamisole was detected in 38% of the hair samples analyzed; its concentration ranged from 0.2 to 0.8 ng/mg. The limit of quantification and limit of detection for levamisole, tropococaine, norcocaine, and cocaine were 0.2 and 0.1 ng/mg, respectively. DI-SPME-GC/MS is a sensitive and specific method to detect the presence of levamisole and cocaine congeners in hair samples.

Changes in Plasma Methionine-Enkephalin Levels Associated With a Cluster Headache Episode

Eighteen male cluster headache (CH) inpatients within a CH series participated in this research. Blood samples were drawn from patients at least 6-hour pain-free after the last acute CH episode and then shortly prior (SP), during, and soon after (SA) a new acute CH attack. Three healthy male, age-comparable drug-free volunteers served as controls; 5 samples were obtained from each of these individual over a 24-hour period. Individual patient's methionine-enkephalin (MET) plasma concentration showed significant changes, and in some subjects, dramatic changes, during the different phases of a single CH episode. Peptide levels followed a general pattern of higher plasma concentration SP to an acute CH attack, followed by decreased levels during the attack itself, and falling even further SA the acute episode. Consistently, 16 of the 18 patients tested showed pre-CH peptide levels significantly higher (arbitrarily the authors considered values 20% or more as "significant") than their own values obtained during the acute CH pain phase, with observed differences reaching 80% or more in 7 of these individuals. For about half of these patients, peptide concentration during the acute CH episode was significantly above the control's range (68.2-87.6 pg MET/mL; control's circulating MET concentration remaining essentially unchanged during a 24-hour period). MET levels were further decreased in essentially all of the post-CH samples, with values falling within (n = 6) or even further below than those in the control's range (n = 11). Neither age, time of CH occurrence, nor patient's use of a number of medications known for failing to influence plasma MET degradation kinetics seemed to significantly influence MET levels. These results might help in the biochemical characterization of the actual phases of a CH episode. Developing drugs modulating MET bioavailability could lead to novel antinociceptive agents useful for the treatment of CH's associated pain.

Decreased Plasma Methionine–Enkephalin Levels in Cluster Headache Patients

Results from a longitudinal study (blood drawn at days 29, 64, 89,124, 142, and 182 of the protocol) shows that the concentration of platelet-poor plasma (PPP) methionine(5)-enkephalin (MET) in healthy, drug-free, white male individuals (n = 5) remains within a relatively narrow range, well within the experimental error of the analytical procedures used. Interindividual differences fail to reach statistical significance [x ± SD and range (MET picograms per mL of PPP) of 91.2 ± 15.1, 67.1-113.5; 69.6 ± 7.5, 66.1-90.1; 76.6 ± 12.6, 58.5-93.1; 86.8 ± 10.9, 76.3-107.4; and 84.5 ± 11.4, 68.9-103.4; for subjects 1-5, respectively]. MET levels were similar to those recorded from single samples obtained from a group of 24 white male, age-comparable, drug-free healthy volunteers [x ± SD and range (picograms of MET per mL of PPP) of 83.3 ± 15.1 and 57.4-119.1]. The controls' range for all the subjects (n = 29) was 57.4-119.1 pgMET/mL PPP. Compared with the controls, individual patients with cluster headache (CH) show a much wider variation in PPP MET levels (blood drawn at different time intervals, at least 10 samples per patient, over a period of 221-298 days), with many (slightly over half) of single values below the controls range; no single MET level was above the controls range [x ± SD and range (picograms of MET per mL of PPP) of 56.4 ± 27.7, 6.1-100.5; 72.6 ± 20.5, 43.0-113.0; 46.0 ± 28.5, 10.0-92.6; 53.6 ± 27.5, 13.0-101.0; 52.0 ± 26.1, 17.5-83.6; 63.5 ± 22.3, 21.7-91.3 for individuals A-F, respectively]. Although interindividual differences within the patients' group were not statistically significant, their peptide levels were significantly lower than those of controls. Neither the presence of unspecified "headaches between clinic visits" and "daily headaches" (patients E and F, respectively), nor the use of a number of drugs known to lack inhibitory activity upon the aminopeptidase-MET degradation reaction, seemed to significantly influence MET concentration. The results could lead to a better understanding of the etiology of the pain associated with CH, with the relative changes in plasma peptide perhaps reflecting the patients' vulnerability to such a condition. Pharmacological modulation of MET function may prove useful in the treatment of CH-associated pain, whether the development of such drugs could find useful pharmacological applications remains to be explored.

Effect of ibuprofen and indomethacin on human plasma melatonin.

Ibuprofen reduced human plasma melatonin (MT) after 2 h when administered orally (400 mg) at 2400 h. Increasing plasma concentrations correlated well with increasing inhibition of serum MT levels during this time. Maximum plasma ibuprofen coincided with minimum plasma MT in 3 out of 4 volunteers. Although two volunteers exhibited a partial recovery in MT levels, concentrations after 6 h were significantly less than 0600 h values in drug-free volunteers. Administration of ibuprofen (400 mg) at 1800 h delayed the nocturnal surge of plasma MT. When a slow release preparation of indomethacin (75 mg) was administered at 1800 h, the dark phase rise of plasma MT was completely prevented. Thus the longer acting cyclooxygenase inhibitor exhibited a longer lasting inhibition of plasma MT concentration.

STIM1 and STIM2 Are Located in the Acidic Ca2+ Stores and Associates with Orai1 upon Depletion of the Acidic Stores in Human Platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

A Validated Method for Simultaneous Screening and Quantification of Twenty-Three Benzodiazepines and Metabolites Plus Zopiclone and Zaleplone in Whole Blood by Liquid-Liquid Extraction and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for detection of 23 benzodiazepines and related compounds in whole blood was developed and validated. The method is used for screening and quantitation of benzodiazepines in whole blood received from autopsy cases and living persons. The detected compounds were alprazolam, bromazepam, brotizolam, chlordiazepoxide, demoxepam, clobazam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, estazolam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, midazolam, nitrazepam, 7-aminonitrazepam, oxazepam, temazepam, triazolam, zaleplon, and zopiclone. Whole blood from drug-free volunteers was used for all experiments. Blood samples (0.200 g) were extracted with ethyl acetate at pH 9. Target drugs were quantified using a Waters ACQUITY UPLC system coupled to a Waters Quattro Premier XE triple quadrupole in positive electrospray ionization, multiple reaction monitoring mode. The use of deuterated internal standards for most compounds verified that the accuracy of the method was not influenced by matrix effects. Extraction recoveries were 73-108% for all analytes. Lower limits of quantification ranged from 0.002 to 0.005 mg/kg. Long-term imprecision (CV%) ranged from 6.0 to 18.7%. We present a fully validated UPLC-MS-MS method for 23 benzodiazepines in whole blood with a run-time of only 5 min and using only 0.200 g of whole blood.

Internal validity of inter-digital web pinching as a model for perceptual diffuse noxious inhibitory controls-induced hypoalgesia in healthy humans

Hot and ice-water immersions are commonly used for heterotopic noxious counter-stimulation (HNCS) in investigations on endogenous pain modulation. However, coincident sympathetic thermoregulatory activity does not allow to differentiate between perceptual hypoalgesia related to baroreflex sensitivity (BRS) or diffuse noxious inhibitory controls (DNIC). The present study analysed the internal validity of another supposedly less confounded tonic pain model (inter-digital web pinching; IWP) regarding its potential as DNIC trigger. We performed a randomized controlled study in 24 healthy gender-matched drug-free volunteers aged 21–54 (median 25) years. The study protocol comprised the assessment of mechanical and thermal perceptual wind-up before and after an IWP (15 N) or hot water immersion trial (HIT; 47.5 °C) of 2 min duration. Wind-up was induced either by 10 repetitive (1 Hz) contact heat (max. 49 °C; 5 × 5 mm thermode) or ballistic impact stimuli (0.5 g at 9 m/s) on the phalanges of the non-dominant hand. Cardiovascular activity, pain experience and corrugator muscle activity were continuously monitored. Although both HNCS forms produced a similar pain experience (45% of scale), a more pronounced cardiovascular activity was observable for the HIT ( P < 0.01). This indicates a higher baroreceptor activity and stronger contamination of painful water immersion by BRS-related hypoalgesia. Regardless of pain modality, wind-up was significantly reduced by HNCS, although this was stronger for painful water immersion than for noxious pinching ( P < 0.01). The HNCS types allow a differentiation between BRS-related and DNIC-like hypoalgesia. IWP proved its validity for DNIC induction, being practically non-confounded by BRS.

Increased basal mechanical pain sensitivity but decreased perceptual wind-up in a human model of relative hypocortisolism

Clinical data have accumulated showing that relative hypocortisolism, which may be regarded as a neuroendocrinological correlate of chronic stress, may be a characteristic of some functional pain syndromes. However, it has not been clarified yet whether deregulations of the hypothalamus–pituitary–adrenal (HPA) axis may directly alter pain perception and thus be causally involved in the pathophysiology of these disorders. To test this hypothesis, we performed a randomized placebo-controlled crossover trial in N = 20 healthy drug-free volunteers (median age 24 yrs) and analyzed the effects of metyrapone-induced hypocortisolism on quantitatively assessed basal mechanical pain sensitivity (1.5–13 m/s impact stimuli), perceptual wind-up (9 m/s impact stimuli at 1 Hz) and temporal summation of pain elicited by inter-digital web pinching (IWP; 10 N pressure stimuli for 2 min). Experimentally induced hypocortisolism significantly decreased pain detection thresholds and augmented temporal summation of IWP-induced pain ( p < .05). The latter effect was dependent on the relative reduction in cortisol levels, and seemed to rely on a potentiated sensitization and not merely on the observed changes in basal pain sensitivity. Perceptual wind-up by contrast was reduced when cortisol synthesis was blocked ( p < .05). This result is reminiscent of findings from animal studies showing a reversal of NMDA receptor activation by glucocorticoid receptor antagonists in neuropathic pain models. Our results speak in favor of a potential causal role of HPA axis alterations in pain chronicity.

Inhibition of Human Plasma Leucine5-Enkephalin Aminopeptidase Hydrolysis by Various Endogenous Peptides and a Select Number of Clinically Used Drugs

We identified a number of clinically used drugs and biologically active endogenous peptides able to significantly decrease the rate of human plasmatic aminopeptidase (AP) leucine-enkephalin (LEU) degradation. Bacitracin, bestatin, fluvoxamine, and each of 4 peptides tested significantly increased, in a dose-dependent manner (10-10 M), LEU degradation half-life (t1/2) in each of 5 plasma samples studied. Each sample was obtained by pooling equal volume of 6 randomly selected, individual plasmas (4 male and 2 female healthy, drug-free volunteers). Thirty subjects (20 females and 10 males) participated in this study. With the exception of fluvoxamine, this inhibitory effect was lacking in various other commonly used drugs with widely different chemical structures and pharmacological profiles, eg, antidepressants (SSRIs, imipramine-like tricyclics, MAOIs), acute antimigraine agents (triptan class drugs), the nonselective beta-adrenergic antagonist propranolol, and serotonin receptor agonists and antagonists. Agents (concentration 10 M used as illustration), listed in decreasing order of LEU-AP inhibitory activity: substance P > angiotensin III > methionine-enkephalin > angiotensin II > fluvoxamine > bestatin gave t1/2 values (+/- SD) of 39.3 +/- 1.1, 29.4 +/- 0.8, 28.3 +/- 0.8, 27.4 +/- 0.7, 24.5 +/- 1.5, and 23.6 +/- 0.9 minutes, respectively. Control, bacitracin, and fluphenazine (known LEU-AP inhibitors were used for comparison) values of 11.8 +/- 1.0, 31.3 +/- 0.7, and 19.6 +/- 1.0 minutes, respectively. As expected, these drugs significantly decreased the initial velocity of peptide degradation; Iv values (+/- SD) of: 0.17 +/- 0.1 (0.02 +/- 0.01), 0.23 +/- 0.2 (0.02 +/- 0.01), 0.25 +/- 0.2 (0.02 +/- 0.01), 0.26 +/- 0.2 (0.03 +/- 0.01), 0.31 +/- 0.1 (0.03 +/- 0.01), and 0.33 +/- 0.1 (0.03 +/- 0.01), respectively; control, bacitracin, and fluphenazine: 1.10 +/- 0.3 (0.12 +/- 0.03), 0.20 +/- 0.1 (0.02 +/- 0.01), and 0.82 +/- 0.2 (0.08 +/- 0.02) pg LEU/min (pg LEU/mg protein/min), respectively. Results emphasize the selective nature of chemical structures required to significantly inhibit AP activity and provided information that could help the rational design of agents with high specificity in a biologic milieu containing multiple peptidases. In this case, targeted modulation of the bioavailability of LEU and other endogenous AP-degraded hormonal and nonhormonal peptides could be useful in the treatment of the pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.

DIFFERENTIAL EFFECTS OF ANTACIDS ON ORAL BIOAVAILABILITY OF TACROLIMUS.

P199 Aims: Catabolism by cytochrome P450 3A4 (CYP3A4) is one of the major determinants of the oral bioavailability of tacrolimus. Cimetidine and omeprazole are known modulators of several CYPs in vitro. In the present study, the impact of cimetidine and omeprazole on oral bioavailability of tacrolimus and on CYP3A4 activity in vivo was examined. Methods: In a historical cohort of 48 (19 females/29 males) renal transplant recipients, who switched standard ulcer prophylaxis with 400 mg of cimetidine to 20 mg of losec, an index of oral bioavailability (BAI = through level of tacrolimus (μg/l), normalized for dose (mg) and weight (kg) and multiplied by 100) was studied. The switch was made at 16-1095 days after transplantation because of epigastric discomfort. Endoscopic evaluation showed reflux oesophagitis in 33 and non complicated gastric/duodenal ulceration in 7 patients. Ulceration was complicated with bleeding in 2 and the remaining 6 patients had a compound of reflux oesophagitis and candidiasis (n=3) or CMV (n=2) or herpes (n=1) infection. For each patient, mean steroid dose, hematocrit, albumin, bilirubin and creatinin during a 5 days’ interval before and after switch were determined. A paired comparison for each parameter before and after switch was made. In addition, the impact of sex, age, days of follow-up, diagnosis and Δ (= value before – after switch) of all determined biological parameters on ΔBAI was studied with multivariate ANOVA. In a cohort of 6 healthy, drug free volunteers, the effect of a 5 days’ course of cimetidine and omeprazole (randomized, cross over design) on intestinal and hepatic CYP3A4/PGP activity was measured by comparing the results of a combined per oral and intravenous 14C-erythromycin breath and urine test after each treatment to baseline values. Results: In the patients, BAI decreased significantly with 15% from 13 to 11 and serumcreatinin decreased significantly with 26% from 2.38 to 1.76 mg/d after switch. Multivariate ANOVA retained a significant but weak (r2=0.16) impact of age on ΔBAI. In the healthy volunteers, a significant increase of intestinal CYP3A4 activity was noticed after treatment with omeprazole, whereas treatment with cimetidine yielded results similar to baseline. Conclusions: Switching antacid therapy from cimetidine to omeprazole in renal transplant recipients is associated with a decrease of dose and weight normalized trough levels of tacrolimus which is independent of corticosteroid dose, time after transplantation, sex, renal function, hematocrit and serumalbumin. This clinical finding can be explained by the increase of intestinal CYP3A4 activity as observed in healthy volunteers after a 5 days’ course of omeprazole.

In vitro Human Plasma Leucine5-Enkephalin Degradation Is Inhibited by a Select Number of Drugs with the Phenothiazine Molecule in Their Chemical Structure

A number of drugs with the phenothiazine molecule in their chemical structure inhibit in a dose-dependent manner human plasmatic aminopeptidase leucine⁵-enkephalin (LEU) metabolism. Half-life peptide degradation was significantly increased by thioridazine > fluphenazine > As-1397 [10-(α-diethylaminopropionyl)phenothiazine] ≧ promethazine ≧ chlorpromazine (final drug conc. 10^–4M); t1/2 (± SD) 21.2 ± 1.1, 19.6 ± 1.0, 17.2 ± 0.9, 17.1 ± 1.0, and 17.1 ± 1.1 min, respectively. Control and bacitracin (known aminopeptidase inhibitor) values were 11.8 ± 1.0 and 31.3 ± 1.7 min, respectively. These drugs significantly decreased (listed in the same order) LEU degradation initial velocity; Iv (± SD) 0.77 ± 0.2, 0.82 ± 0.2, 0.92 ± 0.3, 0.93 ± 0.2, 0.94 ± 0.3 pg LEU/min, respectively. Control and bacitracin 1.10 ± 0.3 and 0.20 ± 0.1 pg LEU/min, respectively. Values represent results from 5 samples, each obtained by pooling 6 individual plasmas (4 male and 2 female; n = 30 healthy, drug-free volunteers). However, neither the phenothiazines ethopropazine, methotrimeprazine, prochlorperazine and trifluoperazine nor the various commonly used heterocyclic antipsychotics tested, e.g., molindone, loxapine, clozapine, haloperidol, sulpiride and thiothixene inhibited plasma LEU degradation kinetics. Our results failed to show correlations between chemical structure, antipsychotic properties and ability to inhibit plasmatic aminopeptidase LEU degradation. Whereas, presence of the phenothiazine molecule appears to be necessary for enzyme inhibition, only five out of nine substituted phenothiazines tested exhibited this effect. Furthermore, there was a lack of correlation between phenothiazines antipsychotic properties and their capacity to inhibit aminopeptidase activity, a property shown by promethazine (antihistaminic) and As-1397 (selective butyrylcholinesterase inhibitor) but lacking in prochlorperazine and trifluoperazine. Our results provide information which could lead to the rational design of agents capable to modulate the bioavailability of enkephalin and other endogenous aminopeptidase-degraded peptides believed to be involved in the etiology and/or pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.

Detection of Cocaine and Its Metabolites in Breast Milk

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.

Susceptibility of PharmChek™ drugs of abuse patch to environmental contamination

The key component of the PharmChek™ sweat patch, the membrane, has been tested for the passage of externally applied materials. Drugs in the uncharged state rapidly penetrated the membrane but charged species were greatly slowed. In basic media, detectable concentrations of cocaine, methamphetamine, and heroin were observed at the earliest collection time (ca. 30 s), after drugs were placed on the outside of the membrane. Drug concentrations increased over the 2 h time course, when amounts detected (1710 ng cocaine, 1060 ng methamphetamine, 550 ng heroin per pad at 2 h) represented 5–17% of the drug deposited on the surface of the sweat patch. Drugs externally applied to human skin were shown to bind readily. Drugs deposited on the skin of drug-free volunteers several days prior to application of the sweat patch were not completely removed by normal hygiene or the cleaning procedures recommended before application of the sweat patch. Even 6 days of normal hygiene did not remove all drugs from externally contaminated skin and positive sweat patches resulted. A mechanism for passage of drugs through the sweat patch membrane, a mechanism for retention of drugs on skin, and a redesign of the sweat patch and modification of its use to reduce external contamination are proposed. Appropriate care should be taken in the interpretation of positive results from a sweat patch test until more research is conducted.

Collagen, Convulxin, and Thrombin Stimulate Aggregation-independent Tyrosine Phosphorylation of CD31 in Platelets: EVIDENCE FOR THE INVOLVEMENT OF Src FAMILY KINASES

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.

PROLONGED EXPOSURE TO BUPIVACAINE INHIBITS WHOLE BLOOD COAGULATION

S277 INTRODUCTION: Epidural and systemically infused local anesthetics decrease the risk for postoperative deep vein thrombosis. [1] We have shown previously that thromboxane A2 (TX) signaling is blocked by prolonged application of local anesthetics. [2] Therefore, we studied if prolonged exposure to bupivacaine prolongs whole blood coagulation as measured by activated clotting time (ACT). METHODS: After IRB approval, blood was obtained from 13 drug-free volunteers and anticoagulated with citrate. Blood samples from the same donor were incubated with buffer, bupivacaine (1 and 10 [micro sign] M), TX receptor antagonist SQ29548 (at supramaximal concentration of 10 [micro sign] M) or bupivacaine + SQ29548 (both at 10 [micro sign] M). Incubation time was 60 min. Anticoagulation was then reversed with CaCl2 and ACT was determined. Values were compared by repeated measures ANOVA and paired t test. RESULTS: Bupivacaine (1 and 10 [micro sign] M) prolonged ACT as compared with control (p<0.05, Figure 1). The two concentrations were equally effective (p=0.58). SQ29548 also prolonged ACT (p<0.05). Bupivacaine prolonged ACT more than did SQ29548 (p<0.05), but equally effective as bupivacaine + SQ29548.Figure 1DISCUSSION: Our data indicate that bupivacaine, at blood concentrations as present during epidural anesthesia significantly interferes with blood clotting. That the two bupivacaine concentrations had equal effect suggests that 1 [micro sign]M bupivacaine is already maximally effective. As bupivacaine was more effective than SQ29548, it is likely to block pathways in addition to TX signaling. This is in agreement with our findings using thrombelastography. [3] Bupivacaine + SQ29548 was no more effective than bupivacaine alone, suggesting that bupivacaine blocks TX signaling effectively. Thus, local anesthetic in blood may play a significant role in the antithrombotic actions of epidural anesthesia.

Topiramate increases brain GABA, homocarnosine, and pyrrolidinone in patients with epilepsy.

To measure the effects of topiramate on brain gamma-aminobutyric acid (GABA) in patients with epilepsy. Topiramate is a new antiepileptic medication with multiple putative mechanisms of action. In a recent meta-analysis of the newer antiepileptic drugs, topiramate was the most potent. Homocarnosine and pyrrolidinone are important metabolites of GABA with antiepileptic actions. In vivo measurements of GABA, homocarnosine, and pyrrolidinone were made of a 14-cm3 volume in the occipital cortex using 1H spectroscopy with a 2.1-Tesla magnetic resonance spectrometer and an 8-cm surface coil. Twelve patients (eight women) with refractory complex partial seizures were studied while using topiramate. Nine epilepsy-free, drug-free volunteers served as control subjects. Topiramate increased mean brain GABA, homocarnosine, and pyrrolidinone concentrations in all patients. In paired measurements, brain GABA increased by 0.7 micromol/g (SD 0.3, n 7, 95% CI 0.4 to 1.0, p < 0.01). Homocarnosine increased by 0.5 micromol/g (SD 0.2, n 7, 95% CI 0.3 to 0.7, p < 0.001). Pyrrolidinone increased by 0.21 micromol/g (SD 0.06, n 7, 95% CI 0.16 to 0.27, p < 0.01). In two additional patients, GABA, homocarnosine, and pyrrolidinone increased after they were switched from vigabatrin to topiramate. Topiramate increased brain GABA, homocarnosine, and pyrrolidinone to levels that could contribute to its potent antiepileptic action in patients with complex partial seizures.

Sensitive and specific method for the determination of josamycin in human plasma by liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of josamycin in human plasma by LC–MS was developed and validated. Josamycin was extracted from human plasma by a single-step liquid–liquid extraction and analyzed by LC–MS via an electrospray ionization interface. Selected ion monitoring was used to detect josamycin and its internal standard. The intra-day precision and accuracy, expressed as C.V. and R.E., ranged from 2.8% to 13.5% and −10.3% to 7.6%, respectively. The lower limit of detection was 0.1 ng/ml and the lower limit of quantitation was set at 1 ng/ml when 0.5 ml of plasma was used. No endogenous interference was observed in human plasma obtained from drug-free volunteers.

Gender-Related Seasonality of Human Platelet Phenolsulfotransferase Activity

This study focused on the investigation of the possible effect of gender and season on the activity of the thermolabile and thermostable forms of platelet phenolsulfotransferase (PST) in a group of 23 healthy, drug-free volunteers of both sexes. The results showed a different seasonal profile of PST activity in men and women: in men, PST seasonal rhythms revealed a shallow profile with higher values in both the spring and summer than in the autumn. Conversely, in women, the PST seasonality showed a profile consisting overall of a main peak in the summer. Also, significant gender-dependent correlations were found between the photoperiod length and PST values. Our findings should stimulate further investigation into gender-dependent molecular mechanisms underlying the regulation of the metabolism of endogenous and xenobiotic agents, such as monoamines and phenols, which are the substrates of PST.