Detection of drugs and hepatitis C virus in used syringes from a needle exchange in Gothenburg, Sweden.

People who inject drugs (PWID) are exposed to serious health risks such as lethal overdoses, addiction and infections. The patterns of drug use and the prevalence of hepatitis C virus (HCV) infection vary greatly between and even within countries. Data on drugs used for injection are important to inform PWID of risks and adapt healthcare. This study aimed to determine which substances are injected in Gothenburg, Sweden, and estimate the risk of HCV transmission. A total of 150 syringes handed in at the needle and syringe exchange program (NEP) in Gothenburg over a week in November 2021 were analysed for drug content using liquid chromatography coupled with high-resolution mass spectrometry. Using a dose-adjusted comparison, the main drug(s) injected was distinguished from the impurities in the syringes containing several drugs. HCV RNA was quantified by real-time PCR in an additional set of 150 syringes. Drugs were detected in >99% of analysed syringes, and the most common drugs were amphetamine (81%), followed by buprenorphine (8.0%), heroin (6.7%) and alprazolam (4.6%). Less common findings were testosterone (2.7%), methylphenidate (2.0%), MDMA (0.7%), trenbolone (0.7%) and zopiclone (0.7%). Eleven syringes (7.3%) contained more than one drug. HCV RNA was detected in 13% of the syringes, and one in 10 contained enough to potentially transmit an infection. This study underlines the importance of access to NEPs for PWID to reduce the risks associated with drug injection.

Retrospective identification of new psychoactive substances in patient samples submitted for clinical drug analysis.

New psychoactive substances (NPS) are life threatening through unpredictable toxicity and limited analytical options for clinicians. We present the retrospective identification of NPS in raw data from a liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based multidrug panel analysis on 14 367 clinical oral fluid samples requested during 2019 mainly by psychiatric and addiction care clinics. Retrospectively analysed NPS included 48 notified originally in 2019 by the European Union Early Warning System (EU EWS) and 28 frequently reported in Sweden. Of 88 included NPS, 34 (mitragynine, flualprazolam, 3F/4F-α-P(i)HP, etizolam, 4F-MDMB-BINACA, cyproheptadine, 5F-MDMB-PICA, isotonitazene, isohexedrone, MDPEP, N-ethylpentedrone, tianeptine, flubromazolam, 4'-methylhexedrone, α-P(i)HP, eutylone, mephedrone, N-ethylhexedrone, 5F-MDMB-PINACA, ADB-BUTINACA, 3-methoxy PCP, 4F-furanylfentanyl, 4F-isobuturylfentanyl, acrylfentanyl, furanylfentanyl, clonazolam, norfludiazepam, 3F-phenmetrazine, 3-MMC, 4-methylpentedrone, BMDP, ethylphenidate, methylone and α-PVP) were identified as 219 findings in 84 patients. Eight NPS notified in 2019 were identified, five before EWS release. NPS occurred in 1.20% of all samples and 1.53% of samples containing traditional drugs, and in 1.87% of all patients and 2.88% of patients using traditional drugs. NPS use was more common in men and polydrug users. Legal (not scheduled) NPS were more used than comparable illegal ones. Retrospective identification could be useful when prioritizing NPS for clinical routine analysis and when studying NPS epidemiology.

Rethinking Drug Analysis in Health Care: High-Throughput Analysis of 71 Drugs of Abuse in Oral Fluid Using Ion Mobility--High-Resolution Mass Spectrometry.

We have identified a clinical need for a sensitive, specific, flexible, comprehensive and affordable analytical technology to efficiently detect polydrug use. In addition, the current standard practice of surveilled urine sampling is uncomfortable for the patient; hence, more patient-friendly sample collection methods are requested. To fill these needs, we have developed and validated a high-throughput liquid chromatography-high-resolution mass spectrometry (LC--HRMS) method for the analysis of drugs of abuse (DoA) in oral fluid (OF). The method covers a panel of 71 substances including traditional DoA, prescription narcotics and new psychoactive substances (NPS), with a guaranteed limit of identification of <3 µg/L for 87% of the analytes. Method validation showed high accuracy (>99.7%), sensitivity (>99.7%) and specificity (100%). Most analytes had a high process efficiency during the salting-out liquid-liquid extraction sample preparation and no or only a minor matrix effect during the analysis. We have implemented this method in clinical routine and present data from 18,579 OF samples collected during routine patient treatment in mainly psychiatric and addiction clinics in West Sweden between September 2020 and June 2021. Seventy-one percent of the samples were positive and a total of 41,472 DoA findings were detected. Amphetamine (27%), buprenorphine (25%), nordiazepam (18%) and alprazolam (16%) were most prevalent. New psychoactive substances were detected in 189 samples (1.0%). The occurrence of polydrug use was common; 34% of the positive samples contained three analytes or more and 12% six or more. To the best of our knowledge, this is the first method for comprehensive analysis of DoA in OF using LC--HRMS and the largest dataset published on the detection of DoA in OF. With the current complex and variable drug use pattern, this broad, cost-effective and reliable method has largely replaced immunoassay screening in urine in our laboratory.

α-Terpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure.

The monoterpene α-terpinene is used as a fragrance compound and is present in different essential oils. It is one of the components responsible for the antioxidant activity of tea tree oil. α-Terpinene is structurally similar to other monoterpenes, e.g., limonene, known to autoxidize on air exposure and form allergenic compounds. The aim of the present study was to investigate the possible autoxidation of α-terpinene at room temperature. To investigate the sensitization potency of air-exposed α-terpinene and the oxidation products formed, the murine local lymph node assay was used. Chemical analysis showed that α-terpinene degrades rapidly, forming allylic epoxides and p-cymene as the major oxidation products and also hydrogen peroxide. Thus, the oxidation pathway differs compared to that of, e.g., limonene, which forms highly allergenic hydroperoxides as the primary oxidation products on autoxidation. The sensitization potency of α-terpinene was increased after air-exposure. The allylic epoxides and a fraction, in which only an α,ß-unsaturated aldehyde could be identified, were shown to be strong sensitizers in the local lymph node assay. Thus, we consider them to be the major contributors to the increased sensitization potency of the autoxidized mixture. We also investigated the presence of α-terpinene and its oxidation products in four different tea tree oil samples of various ages. α-Terpinene and its oxidation products were identified in all of the tea tree oil samples. Thus, from a technical perspective, α-terpinene is a true antioxidant since it autoxidizes rapidly compared with many other compounds, preventing these from degradation. However, as it easily autoxidizes to form allergens, its suitability can be questioned when used in products for topical applications, e.g., in tea tree oil but also in cosmetics and skin care products.

Bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant.

In the present work, the characterization of the biotransformation and bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant (Acomplia) is described. Rimonabant was approved in Europe in 2006 for the treatment of obesity but was withdrawn in 2008 because of a significant drug-related risk of serious psychiatric disorders. The aim of the present work is to characterize the biotransformation and potential bioactivation pathways of rimonabant in vitro in human and rat liver microsomes. The observation of a major iminium ion metabolite led us to perform reactive metabolite trapping, covalent binding to proteins, and time-dependent inhibition of cytochrome P450 3A4 studies. The major biotransformation pathways were oxidative dehydrogenation of the piperidinyl ring to an iminium ion, hydroxylation of the 3 position of the piperidinyl ring, and cleavage of the amide linkage. In coincubations with potassium cyanide, three cyanide adducts were detected. A high level of covalent binding of rimonabant in human liver microsomes was observed (920 pmol equivalents/mg protein). In coincubations with potassium cyanide and methoxylamine, the covalent binding was reduced by approximately 40 and 30%, respectively, whereas GSH had no significant effect on covalent binding levels. Rimonabant was also found to inhibit cytochrome P450 3A4 irreversibly in a time-dependent manner. In view of these findings, it is noteworthy that, to date, no toxicity findings related to the formation of reactive metabolites from rimonabant have been reported.

Diphenylthiourea, a common rubber chemical, is bioactivated to potent skin sensitizers.

Diphenylthiourea (DPTU) is a known skin sensitizer commonly used as a vulcanization accelerator in the production of synthetic rubber, for example, neoprene. The versatile usage of neoprene is due to the multifaceted properties of the material; for example, it is stretchable, waterproof, and chemical- and abrasion-resistant. The wide application of neoprene has resulted in numerous case reports of dermatitis patients allergic to DPTU. The mechanism by which DPTU works as a contact allergen has not been described; thus, the aim of the present study was to investigate if DPTU is a prohapten that can be activated by skin metabolism. The metabolic activation and covalent binding of (14)C-labeled DPTU to proteins were tested using a skinlike cytochrome P450 (P450) cocktail containing the five most abundant P450s found in human skin (CYP1A1, 1B1, 2B6, 2E1, and 3A5) and human liver microsomes. The incubations were carried out in the presence or absence of the metabolite trapping agents glutathione, methoxylamine, and benzylamine. The metabolism mixtures were analyzed by LC-radiochromatography, LC-MS, and LC-MS/MS. DPTU was mainly metabolically activated to reactive sulfoxides resulting in desulfurated adducts in both enzymatic systems used. Also, phenylisothiocyanate and phenylisocyanate were found to be metabolites of DPTU. The sensitizing capacity of the substrate (DPTU) and three metabolites was tested in the murine local lymph node assay. Two out of three metabolites tested were strong skin sensitizers, whereas DPTU itself, as previously known, was negative using this mouse model. In conclusion, DPTU forms highly reactive metabolites upon bioactivation by enzymes present in the skin. These metabolites are able to induce skin sensitization and are probable causes for DPTU allergy. To increase the possibilities of diagnosing contact allergy to DPTU-containing items, we suggest that suitable metabolites of DPTU should be used for screening testing.

Cutaneous metabolic activation of carvoxime, a self-activating, skin-sensitizing prohapten.

Bioactivation of low molecular weight compounds in the skin can cause contact sensitization. We have previously shown that the alpha, beta-R-unsaturated oxime R-carvoxime [1, (R)-2-methyl-5-isopropenylcyclohex-2-enone oxime] is bioactivated to two diastereomeric highly reactive and strongly sensitizing alpha, beta-epoxy oxime metabolites. To investigate if this metabolic activation is catalyzed by the major cytochrome P450 (P450) enzymes found in human skin, incubations of 1 with a skinlike P450 cocktail in the presence of glutathione were carried out. We identified three glutathione conjugates in the incubation mixture arising from two diasteomeric alpha, beta-epoxy oxime metabolites of 1, thus showing that the metabolic activation of 1 is P450-mediated. A P450 identification study using the individual P450 enzymes present in the skinlike P450 cocktail showed the involvement of P450 1A1 and 1B1 and also to some extent 2B6. P450 1B1 metabolism of 1 was found to be stereoselective as glutathione conjugates from only one of the alpha, beta-epoxyoxime metabolites were identified (metabolite 2). Additionally, 1 was found to be an inducer of P450 1B1 (but not 1A1) in human monocyte-derived dendritic cells (moDCs) and to some extent in normal human epidermal keratinocytes. A further transcriptional gene expression change observed in moDCs was a 44-fold upregulation of IL-8, a marker often used for assessment of sensitizing potential of contact allergens. The autoinduction of P450 1B1 by 1 may be a key event in the development of contact allergy to 1 and may also explain why only metabolite 2, and not 3, was found to elicit an allergic response in mice sensitized to 1. Our data show that the alpha, beta-unsaturated oxime 1 is bioactivated by human cutaneous P450, thus forming highly allergenic metabolites, and has the potential to induce its own bioactivation pathway, particularly in antigen-presenting cells.

Oximes: metabolic activation and structure-allergenic activity relationships.

Metabolic activation of chemicals (prohaptens) in the skin can cause allergic contact dermatitis. We have explored structure-allergenic activity relationships for seven potential oxime prohaptens using the local lymph node assay and a GSH trapping screen with liver microsomes. The general structure-allergenic activity relationships found were that an alpha,beta-unsaturation is necessary for an oxime to be a prohapten and that increased steric hindrance around this double bond leads to reduction in sensitizing capacity. We also found that sensitizing oximes can be distinguished in vitro from nonsensitizers by monitoring of mono-oxidized (+16 Da) GSH conjugates in the GSH trapping screen. However, care should be taken when interpreting data from GSH trapping screens, as nonsensitizers may also form GSH conjugates via alternative mechanisms. This investigation emphasizes the importance of considering cutaneous bioactivation in toxicity assessment of chemicals used in contact with the skin.

Allergic contact dermatitis--formation, structural requirements, and reactivity of skin sensitizers.

Contact allergy is caused by a wide range of chemicals after skin contact. Its clinical manifestation, allergic contact dermatitis (ACD), is developed upon repeated contact with the allergen. This perspective focuses on two areas that have yielded new useful information during the last 20 years: (i) structure-activity relationship (SAR) studies of contact allergy based on the concept of hapten-protein binding and (ii) mechanistic investigations regarding activation of nonsensitizing compounds to contact allergens by air oxidation or skin metabolism. The second area is more thoroughly reviewed since the full picture has previously not been published. Prediction of the sensitizing capacity of a chemical is important to avoid outbreaks of ACD in the population. Much research has been devoted to the development of in vitro and in silico predictive testing methods. Today, no method exists that is sensitive enough to detect weak allergens and that is robust enough to be used for routine screening. To cause sensitization, a chemical must bind to macromolecules (proteins) in the skin. Expert systems containing information about the relationship between the chemical structure and the ability of chemicals to haptenate proteins are available. However, few designed SAR studies based on mechanistic investigations of prohaptens have been published. Many compounds are not allergenic themselves but are activated in the skin (e.g., metabolically) or before skin contact (e.g., via air oxidation) to form skin sensitizers. Thus, more basic research is needed on the chemical reactions involved in the antigen formation and the immunological mechanisms. The clinical importance of air oxidation to activate nonallergenic compounds has been demonstrated. Oxidized fragrance terpenes, in contrast to the pure terpenes, gave positive patch test reactions in consecutive dermatitis patients as frequently as the most common standard allergens. This shows the importance of using compounds to which people are exposed when screening for ACD in dermatology clinics.

Metabolic epoxidation of an alpha,beta-unsaturated oxime generates sensitizers of extreme potency. Are nitroso intermediates responsible?

Metabolic activation of inherently nonprotein-reactive compounds (prohaptens) in the skin can lead to development of contact allergy, a chronic skin disease. The prohapten hypothesis has existed for more than 20 years; yet, detailed knowledge regarding the mechanisms of activation as well as what structural moieties can be transformed to protein-reactive sensitizers is still limited. Today, the consideration of cutaneous bioactivation is important when developing nonanimal-based assays for prediction of contact allergenic activity, as only methods that include skin metabolism are able to detect prohaptens as sensitizers. We have studied the mechanism of activation of the prohapten carvoxime (1), a strongly sensitizing but in itself poorly protein-reactive alpha,beta-unsaturated oxime. alpha,beta-Unsaturated oximes represent a novel class of prohaptens, which previously have never been investigated for potential metabolic activation. To identify reactive metabolites formed from 1, liver microsomal incubations in the presence of glutathione were carried out. Putative reactive metabolites were synthesized, and their allergenic activity, chemical reactivity toward nucleophiles, and ability to elicit a contact allergenic response in animals induced with 1 were assessed. We found that 1 is metabolically activated by epoxidation of the allylic carbon-carbon double bond. The alpha,beta-epoxy oxime metabolites were found to be sensitizers of extreme potency in the local lymph node assay and highly reactive toward nucleophilic amino acids and a model peptide. One of the two diastereomeric epoxy metabolites also elicited an allergic reaction in mice sensitized to 1, in the mouse ear swelling test. Furthermore, this study presents strong indications that the basis of the high reactivity and sensitizing capacity observed for the alpha,beta-unsaturated oximes is related to their ability to form highly reactive nitroso intermediates by tautomerization. To our knowledge, the formation of nitrosoalkenes by oxidative metabolism of alpha,beta-unsaturated oximes has not been shown so far.

A skin-like cytochrome P450 cocktail activates prohaptens to contact allergenic metabolites.

Allergic contact dermatitis is a complex syndrome representing immunological responses to cutaneous exposure to protein-reactive chemicals. Although many contact sensitizers directly can elicit this disorder, others (prohaptens) require activation. Knowledge regarding the activating mechanisms remains limited, but one possibility is metabolic activation by cytochrome P450 (CYP) enzymes in the skin. We have, after quantitative reverse transcriptase-PCR studies of the CYP content in 18 human skin samples, developed an enriched skin-like recombinant human (rh) CYP cocktail using CYP1A1, 1B1, 2B6, 2E1, and 3A5. To validate the rhCYP cocktail, a prohaptenic conjugated diene ((5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene) was investigated using: the skin-like rhCYP cocktail, a liver-like rhCYP cocktail, single rhCYP enzymes, liver microsomes, keratinocytes, and a dendritic cell (DC) assay. The diene was activated to sensitizing epoxides in all non-cell-based incubations including the skin-like rhCYP cocktail. An exocyclic epoxide metabolite ((7R)-7-isopropenyl-4-methyl-1-oxaspiro[2.5]oct-4-ene) was found to be mainly responsible for the allergenic activity of the diene. This epoxide also induced pronounced DC activation indicated by upregulation of IL-8. The skin-like rhCYP cocktail provides a simplified alternative to using skin tissue preparations in mechanistic studies of CYP-mediated skin metabolism of prohaptens and offers the future possibility of designing in vitro predictive assays for assessment of allergenic activity of prohaptens.

Conjugated dienes as prohaptens in contact allergy: in vivo and in vitro studies of structure-activity relationships, sensitizing capacity, and metabolic activation.

There is a great interest in developing in vitro/in silico methods for the prediction of contact allergenic activity. However, many proposed methods do not take the activation of prohaptens to sensitizers by skin metabolism into account. As a consequence, consumer products containing potent sensitizers could be marketed. To identify prohaptens, studies regarding their structure-activity relationships and the mechanisms of their activation must be conducted. In the present investigation, we have studied the structure-activity relationships for alkene prohaptens. A series of seven alkenes (1-7), all of the same basic structure but with variation in the number and position(s) of the double bond(s), were designed and screened for sensitizing capacity using the murine local lymph node assay. Compounds 1-7 were also incubated with liver microsomes in the presence of glutathione to trap and identify reactive metabolites. The metabolic conversion of three alkenes (9-11) to epoxides (12-15) was also studied along with comparison of their sensitizing capacity. Our results show that conjugated dienes in or in conjunction with a six-membered ring are prohaptens that can be metabolically activated to epoxides and conjugated with GSH. Related alkenes containing isolated double bonds and an acyclic conjugated diene were shown to be weak or nonsensitizers. For the first time, the naturally occurring monoterpenes alpha-phellandrene, beta-phellandrene, and alpha-terpinene were demonstrated to be prohaptens able to induce contact allergy. The difference in sensitizing capacity of conjugated dienes as compared to alkenes with isolated double bonds was found to be due to the high reactivity and sensitizing capacity of the allylic epoxides metabolically formed from conjugated dienes. We recommend that these structure-activity relationship rules are incorporated into in silico predictive databases and propose that the prediction of contact allergenic activity of suspected prohaptens is based on assessment of susceptibility to metabolic activation and chemical reactivity of potential metabolites.

An alpha,beta-unsaturated oxime identified as a strong contact allergen. Indications of antigen formation via several pathways.

The aim of this study was to examine the possible skin sensitizing effect of oximes, employing an alpha,beta-unsaturated oxime as the model compound. Oximes are not frequently used as biologically active compounds. However, they have been shown to possess both anti-inflammatory and anti-allergic activities. Furthermore, in a recent study, a number of oximes and oxime-ethers of hydroxylated benzaldehydes and acetophenones were found to be powerful antioxidants suggested to be used in consumer products such as cosmetics and food. Although there are only few reports on the sensitizing effect of oximes, their ability to be hydrolyzed to the corresponding ketones or aldehydes makes them potential contact allergens. The oxime investigated in this study was demonstrated to be a strong contact allergen in both mice and guinea pigs, capable of sensitize the control animals after only one dermal exposure. In order to elucidate the mechanisms for the formation of the complete antigen, a variety of analogues with different reactivity were tested. The results indicate that alpha,beta-unsaturated oximes can react with proteins via several different pathways. Most likely, a metabolic transformation is involved. Due to the strong allergenic effect of the oxime investigated, we strongly advise against the use of such oximes in consumer products until a better understanding of their interactions with biological macromolecules has been obtained.

A conjugated diene identified as a prohapten: contact allergenic activity and chemical reactivity of proposed epoxide metabolites.

A hapten causing allergic contact dermatitis binds covalently to macromolecules via nucleophilic-electrophilic reactions or radical couplings. A prohapten can be seen as a chemically inert compound without electrophilic or radical forming properties. To exert its activity, the prohapten is activated, for example, metabolically, to the hapten. We have investigated the contact allergenic properties of a diene, (5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene (1), as a potential prohapten, and we found it to be a sensitizer in animal studies. The activity is likely to be exerted via epoxide metabolites. Thus, two potential metabolites of the investigated diene, (4S)-1,2-epoxy-4-isopropenyl-1-methyl-6-methylene-cyclohexane (3) and (7R)-7-isopropenyl-4-methyl-1-oxa-spiro[2.5]oct-4-ene (4), were synthesized and subjected to animal tests. Both epoxides were sensitizers. They also elicited significant reactions when tested in animals induced with 1, which indicates that they are formed from the diene in the skin. Furthermore, incubation of 1 with human liver microsomes produced both epoxides. The chemical reactivity of 1, 3, and 4 was investigated in relation to a hexapeptide, H-Pro-His-Cys-Lys-Arg-Met-OH. No adducts were obtained from reactions between the peptide and 1. However, epoxide 3 bound covalently to the cysteine residue and epoxide 4 to both the cysteine and proline residues. Since it is possible to relate the sensitizing capacity of a compound to its key physicochemical properties, knowledge-based expert systems have been developed to predict the toxicity of novel compounds by comparing the structure with activity data stored in the computer database. A diene related to 1 found in the knowledge-based expert system DEREK was considered as a nonsensitizer by this system. Our study indicates that conjugated dienes can be metabolized to contact allergens in the skin. Thus, when constructing predictive test methods based on SARs, it is important to analyze not only the virtual chemical structure of a compound but also its ability to act as a prohapten.