Metabolism and toxicological analysis of synthetic cannabinoids in biological fluids and tissues.

Synthetic cannabinoids, which began proliferating in the United States in 2009, have gone through numerous iterations of modification to their chemical structures. More recent generations of compounds have been associated with significant adverse outcomes following use, including cognitive and psychomotor impairment, seizures, psychosis, tissue injury and death. These effects increase the urgency for forensic and public health laboratories to develop methods for the detection and identification of novel substances, and apply these to the determination of their metabolism and disposition in biological samples. This comprehensive review describes the history of the appearance of the drugs in the United States, discusses the naming conventions emerging to designate new structures, and describes the most prominent new compounds linked to the adverse effects now associated with their use. We review in depth the metabolic pathways that have been elucidated for the major members of each of the prevalent synthetic cannabinoid drug subclasses, the enzyme systems responsible for their metabolism, and the use of in silico approaches to assist in predicting and identifying the metabolites of novel compounds and drug subclasses that will continue to appear. Finally, we review and critique analytical methods applied to the detection of the drugs and their metabolites, including immunoassay screening, and liquid chromatography mass spectrometry confirmatory techniques applied to urine, serum, whole blood, oral fluid, hair, and tissues.

Pharmacology, Toxicology, and Adverse Effects of Synthetic Cannabinoid Drugs.

Synthetic cannabinoid drugs have become an established part of the recreational drug landscape in the United States and internationally. These drugs are manufactured in clandestine laboratories internationally and distributed in the United States in smoking mixtures, use of which produces effects very similar to use of marijuana. The adverse-effect profile of the drugs has not been studied in humans and infrequently in animal models, so much of the information about their toxicity comes from emergency department and treatment reports and forensic case studies. This review considers the discovery and characterization of the endocannabinoid system, approaches to receptor-binding studies of various synthetic cannabinoids from the first wave of naphthoylindoles (e.g., JWH-018) to the emerging adamantoylindole drugs (e.g., AKB-48), and their analogs, to evaluate the potential activity of drugs in this class. Currently employed approaches to assessing functional activity of the drugs using in vitro and in vivo models is also described, and comparisons made to the effects of THC. The physiological effects of activation of the endocannabinoid system in humans are reviewed, and the physiological effects of cannabinoid use are described. Case reports of adverse events including emergency department admissions, mental health admissions, and clinical and forensic case reports are presented in detail and discussed to summarize the current state of knowledge of adverse effects, both clinical and forensic in humans, including effects on driving ability, and tissue injury and death. The greatest weight is accorded to those reports that include toxicological confirmation of use. Finally, we discuss the current status of attempts to schedule and control the distribution of synthetic cannabinoids and the relevance of receptor binding and functional activity in this context. There is growing toxicological and pharmacological evidence of impairment, psychosis, tissue injury, and isolated deaths attributable to this emerging class of drugs.

The use of vitreous humor as an alternative to whole blood for the analysis of benzodiazepines.

In postmortem drug analysis, the most commonly used sample matrix is whole blood. However, postmortem changes can denature this matrix, resulting in a loss or degradation of drugs, thus biasing analytical findings. Vitreous humor is thought to be less affected by these changes and should, therefore, have the potential to provide a more reliable estimation of antemortem drug concentrations. To assess the usefulness of vitreous humor for the analysis of benzodiazepine drugs, vitreous humor and whole blood were obtained postmortem in 27 cases. Three benzodiazepine drugs were investigated-temazepam, diazepam, and desmethyldiazepam. For temazepam and diazepam, some correlation was found between the matrices (R2 = 0.789 and 0.724, respectively). However, for desmethyldiazepam, no correlation was observed (R2 = 0.068). Regression analysis on plots of vitreous humor versus blood concentrations produced gradients of less than 1.0 showing that, in general, levels in blood are higher than the corresponding levels in vitreous humor.

Hair analysis for pharmaceutical drugs. II. Effective extraction and determination of sildenafil (Viagra) and its N-desmethyl metabolite in rat and human hair by GC-MS.

In order to study the incorporation of sildenafil (SDF) and its N-demethylated metabolite (norSDF) into hair, animal model experiments were carried out. After shaving the back hair, SDF was dosed to two sets of three male dark-agouti pigmented rats (5 weeks old) per each group at 25 mg/kg once a day for 5 successive days with intraperitoneal (i.p.) (set1) and oral administration (set2). The regrown back hair was collected 14 d after the first administration. Three typical extraction methods, using methanol-5 M hydrochloric acid, methanol-trifluoroacetic acid and 1 M sodium hydroxide, were evaluated using the rat hair samples containing SDF and norSDF. Methanol-5 M hydrochloric acid was the best extraction method in terms of high efficiency and reproducibility. The extract was purified using Bond Elut Certify columns and was derivatized with trimethylsilylimidazole: N,O-bis(trimethylsilyltrifluoroacetamide): trimethylchlorosilane (3: 3: 2) at 90 degrees C for 30 min. The trimethylsilylated products were analyzed by GC-MS using selected ion monitoring. SDF and norSDF were simultaneously detected in the rat hair. The hair concentrations were 4.9-6.3 (av. 5.8) ng/mg and 15.6-20.3 (av. 17.6) ng/mg for SDF and norSDF, respectively, with i.p. administration, and 2.6-4.1 (av. 3.6) ng/mg and 8.1-10.4 (av. 9.1) ng/mg with oral administration. The hair concentrations of norSDF were about three times higher than those of SDF, and the ratios of both compounds showed no significant difference between i.p. and oral administrations. This method was applied to the scalp hair of two patients who orally took SDF at regular intervals for the treatment of penile erectile dysfunction. The hair concentrations of SDF and norSDF in the two patients were 19.8 and 55.9 ng/mg, and 1.7 and 5.6 ng/mg, respectively.

Hair analysis: self-reported use of "speed" and "ecstasy" compared with laboratory findings.

Drug use histories were collected from 100 subjects recruited from the "dance scene" in and around Glasgow, Scotland. In addition, each subject donated a hair sample which was analyzed by gas chromatography/mass spectrometry (GC/MS) for amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MD MA) and 3,4-methylenedioxyethylamphetamine (MDEA). The hair samples were analyzed in two 6 cm segments or in full, ranging from 1.5 to 12 cm depending on the length of the hair. Approximately 10 mg of hair was ground to a fine powder before treatment with beta-glucuronidase/aryl sulfatase. A solid-phase extraction procedure was carried out followed by derivatization with pentafluoropropionic anhydride (PFPA). All extracts were analyzed by gas chromatography/mass spectrometry (GC/MS). Of the 139 segments analyzed, 77 (52.5%) were positive for at least one of the five amphetamines. The drug concentrations found in the hair were compared with the self-reported drug histories. A concordance of greater than 50% was found between the self-report data and levels detected in hair. However, no correlation was found between the reported number of "ecstasy" tablets consumed and the drug levels detected in hair. An increase in the average drug levels measured was observed from low to high use (number of "ecstasy" tablets/month). A large number of false negatives and a low number of false positives were observed.

Hair testing for "ecstasy" (MDMA) in volunteer Scottish drug users.

The aim of the study was to compare self reported "ecstasy" use with the results of the analysis of hair harvested from the same users. Subjects were recruited by multisite chain-referral sampling within the 1994-95 "dance scene" in Glasgow. One hundred subjects donated hair after completing a lengthy interviewer-administered questionnaire. Overall gross concordance between self reported "ecstasy" use and discovery of MDMA (or related compounds) in analysed hair did not surpass 59%, and no relationship had a Cohen's kappa of more than 0.08. Within the positive concordant dataset (n = 52), scatter was considerable, with no correlation being significant, and none more strongly positive than -0.0518. The results presented here indicate that, as far as MDMA is concerned, if judged by self-report, hair does not reach a level of apparent accuracy that would permit its use as a general population estimator. However, hair testing is probably more reliable than self-report, and its accuracy could be verified independently if large-scale inter- and intra-laboratory comparative research is conducted.

Comparison of solid-phase extraction and supercritical fluid extraction for the analysis of morphine in whole blood.

A comparative study of the quantitative determination of morphine in whole blood using solid-phase extraction (SPE) and supercritical fluid extraction (SFE) is described. Comparative studies were made of the two techniques for the extraction of morphine from authentic forensic blood specimens. Quantitative results indicate that morphine levels measured using SPE correspond well to morphine levels produced using SFE. The two techniques are therefore comparable, although SFE is faster and cleaner and extracts may be produced with higher analyte recoveries than with SPE. This paper presents a comparison of the two techniques and the morphine concentrations determined in blood.

Vitreous humor as an alternative sample to blood for the supercritical fluid extraction of morphine and 6-monoacetylmorphine.

The use of vitreous humor as an alternative sample to blood was investigated for the detection of heroin abuse by quantifying levels of morphine and 6-monoacetylmorphine (6-MAM) in post-mortem samples. The levels achieved in each of the two toxicological specimens were compared on a case-to-case basis to determine if a correlation existed. A total of 20 positive morphine cases were examined. In general, the levels of morphine in blood were higher than in the corresponding vitreous humor samples, with some correlation existing. 6-MAM was found in 15 blood samples and 17 vitreous humor samples. Although no correlation was found between the levels of 6-MAM in blood and vitreous humor, the latter may still be used for verification of heroin abuse.

Development of a supercritical fluid extraction method for the determination of temazepam in whole blood.

A supercritical fluid extraction (SFE) procedure for the analysis of temazepam from whole blood was developed. Quantitative recoveries were obtained by high-performance liquid chromatography using prazepam as an internal standard and carefully monitoring the extraction temperature and pressure. The results were found to compare well with those obtained by solid-phase extraction techniques, but they also had the advantages of reduced solvent consumption and minimal sample handling. The application of this method to authentic forensic blood specimens showed the SFE method to be useful as an alternative procedure for the extraction of temazepam in the toxicology laboratory.

Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes.

The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effects of the gypsy retrotransposon by blocking enhancer activity. These repressive effects are general, can occur over long distances and require that the su(Hw) protein is bound between the affected enhancer and promoter. The effects of the su(Hw) binding region on yolk protein (yp) gene expression were determined. These genes are regulated by shared enhancers in the intergenic region, which provided a method to examine whether an enhancer blocked by the su(Hw) protein remained functional. We demonstrate that a blocked enhancer is completely active, supporting the proposal that the su(Hw) protein is an insulator protein that acts by forming a new boundary in a pre-existing chromatin domain, thereby preventing the interaction of regulatory elements located upstream of the insertion site with the promoter. In addition, we found that yp promoter function is not diminished by sharing enhancers, suggesting that these enhancers are not rate limiting for transcriptional activation. Lastly, our data indicate that yp promoter activity is interdependent, such that transcription from one promoter influences the level of activity of the linked promoter.

Cold- and cryopreservation of human liver and kidney slices.

Tissue slices may provide a rapid and economical way of determining cold ischemic effects on human liver and kidney cell viability and metabolism. In contrast to isolated hepatocyte cultures, tissue slices offer an in vitro system which more closely resembles the in vivo situation because of the differentiation and functional heterogeneity of the slice. In this study, human liver and kidney slices were cold stored for 10 days in Belzers University of Wisconsin (UW), Euro-Collins, and Modified Sacks solutions. Another set of slices was cryopreserved at 1 degree C/min for liver and 12 degrees C/min for kidney using a 10% dimethyl sulfoxide/fetal calf serum (FCS) cryoprotectant solution. The cold- and cryopreserved slices were incubated in roller culture for 4 h using FCS as the media. Liver slice viability was assessed by K+ content, protein synthesis, gluconeogenesis, and urea synthesis. Kidney slice viability was assessed using K+ content, protein synthesis, and organic ion transport (PAH and TEA). Human kidney slices were cold preserved in UW for 4-6 days, while the human liver slices were preserved for 12-24 h depending on the viability parameter. Following cryopreservation, human liver slice viability was retained at between 65 and 90% of control values, while kidney slice viability was maintained between 70 and 90% of control values depending on the viability parameter. These results indicate that this human in vitro tissue slice system can be used to optimize preservation solutions and methods. The ability to cold- and cryopreserve human slices could facilitate the more efficient utilization of human tissue.

Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats.

The goal of this study is to determine whether the previously observed, short-term protective effect of estrogen and diphosphonate compounds against osteopenia in ovariectomized (OVX) rats can be maintained for an entire year. Sham-operated control and OVX rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and risedronate (NE-58095) for 360 days after surgery. Their proximal tibiae and first lumbar vertebrae were processed undecalcified for quantitative bone histomorphometry. Both skeletal sites in vehicle-treated OVX rats were characterized by decreased cancellous bone volume and increases in most cellular and fluorochrome-based indices of bone formation and resorption. Treatment of OVX rats with estrogen or diphosphonate compounds depressed bone turnover and provided nearly complete protection against cancellous bone loss. Long-term EHDP treatment induced a moderate mineralization defect, as indicated by increased absolute osteoid volume and a high proportion of osteoid surfaces devoid of adjacent osteoblasts. In contrast, NE-58095 had minimal effects on bone mineralization. These findings indicate that diphosphonate compounds and estrogen provide long-term protection against tibial and vertebral osteopenia in OVX rats. They further indicate that diphosphonate compounds merit consideration as an alternative to estrogen for the prevention of postmenopausal bone loss.

Long-term effects of ovariectomy and aging on the rat skeleton.

The long-term skeletal effects of ovariectomy and aging were studied in female Sprague-Dawley rats sacrificed at 270, 370, and 540 days after bilateral ovariectomy (OVX) or sham surgery at 90 days of age. The proximal tibia was processed undecalcified for quantitative bone histomorphometry. For continuity, data from these late time points were combined with previously published data from earlier time points (0-180 days). A biphasic pattern of cancellous bone loss was detected in the proximal tibial metaphysis of OVX rats. An initial, rapid phase of bone loss out to 100 days was followed by an intermediate period of relative stabilization of cancellous bone volume at the markedly osteopenic level of 5-7%. After 270 days, a slow phase of bone loss occurred during which cancellous bone volume declined to 1-2%. Both the initial, rapid phase and the late, slow phase of bone loss in OVX rats were associated with increased bone turnover. In control rats, cancellous bone volume remained constant at 25-30% out to 270 days (12 months of age), then decreased to approximately 10% by 540 days (21 months of age). This age-related bone loss was also associated with increased bone turnover. It is interesting to note that the proximal tibial growth plates were closed in approximately a quarter of the control rats by 15-21 months of age. Our data indicate that a slow rate of bone loss and increased bone turnover persist in OVX rats during the later stages of estrogen deficiency. Therefore, the development of osteopenia is coincident with increased bone turnover in OVX rats as well as in aged, control rats.

Endocrine and pharmacological suppressors of bone turnover protect against osteopenia in ovariectomized rats.

This study was designed to test the hypothesis that endocrine and pharmacological suppressors of bone turnover prevent the development of osteopenia during estrogen deficiency. Sham-operated control and ovariectomized (OVX) rats were treated intermittently with vehicle alone, estrogen, or the diphosphonate compounds etidronate disodium (EHDP) and NE-58095 [2-(3-pyridinyl)2-hydroxyethylidene-1,1-bisphosphonate disodium] for 35 or 70 days after surgery. Their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. Vehicle-treated OVX rats were characterized by decreased cancellous bone volume and 3- to 4-fold increases in osteoblast surface, osteoclast surface, bone formation rate, and bone resorption rate. Treatment of OVX rats with estrogen and NE-58095 provided complete protection against bone loss and significantly depressed all of the above indices of bone turnover. OVX rats treated with EHDP exhibited at least partial protection against bone loss and decreased bone turnover. EHDP induced a mild mineralization defect, as indicated by a prolonged mineralization lag time at the tibial endocortical surface. The new diphosphonate compound NE-58095 did not impair bone mineralization. Our results indicate that endocrine and pharmacological suppressors of bone turnover prevent the development of osteopenia during the early stages of estrogen deficiency. If confirmed by clinical trials in humans, diphosphonate compounds may prove to be an alternative to estrogen for the prevention of postmenopausal bone loss.