The Metabolism and Detection of New Psychoactive Substances and Emerging Drug Threats in a Poly-drug Context

Lead Research Organisation: University of Dundee
Department Name: Ctr for Anatomy & Human Identification


Context of the project:

934 drug related deaths were recorded in Scotland in 2017, the highest rate reported in Europe and nearly 3 times that recorded in England and Wales. Almost all deaths involved poly-drug abuse and the most common substances involved are opiates and opioids (e.g. heroin and methadone); benzodiazepines (e.g. etizolam and diazepam) and gabapentinoid drugs (gabapentin and pregabalin). Drug users state that gabapentanoids are taken prior to heroin (a mixture of paracetamol, caffeine and diamorphine) to heighten and prolong the effects of the heroin. The mechanism of this reported effect is currently unknown. Users will also take large amounts, often up to 10-30 tablets at a time, of 'street diazepam'. These are illicitly manufactured tablets containing a wide variety of benzodiazepine drugs, often novel drug analogues unlicensed for use in the UK and which are a unique feature of the illicit drug market in Scotland. The drug present, its concentration and its potency will vary widely over time, meaning that users are involved in a form of 'Russian roulette' with the drugs they are taking. When users are tested for drugs using a standard multi-panel drug test field urine test device, they often provide a negative urine test result for benzodiazepines even though the client claims to have recently consumed 'street diazepam' tablets suggesting poor cross reactivity with novel compounds.

Aims and Objectives:

As benzodiazepines are implicated in so many drug related deaths the project also investigates the development of rapid field test methods to distinguish between the different types of benzodiazepines in 'street diazepam' tablets utilising both classical colorimetric and novel technological solutions. It will also assess the limits of detection and selectivity of commonly used urine test kits for

The aim of this project is to use advanced mass spectrometry detection techniques (quadrupole time of flight (QToF-MS/MS) and triple quadrupole (QQQ-MS/MS) tandem mass spectrometry instruments to investigate the metabolism, co-metabolism or inhibition of metabolism of commonly abused drugs to assess the metabolic impact of co-ingestion of these substances on users. The analytical techniques are developed to determine if specific toxic metabolites are formed and the project will validate measurement methods for their detection if present.

Novel physical sciences/engineering aspects of the project

Advanced mass spectrometry is normally applied in the pharmaceutical industry and drug discovery to investigate drug interactions between legal substances which might be co-prescribed or ingested but is rarely applied to the study of drug interactions between pharmaceutically produced drugs and illicitly produced novel drugs of the types in this study. It will adapt and optimise the existing methodologies to new applications.

There are currently no commercially available field tests to rapidly distinguish between closely benzodiazepine drugs and the urine test kits currently being used by front-line staff are not fully validated for the detection of novel benzodiazepines. This project aims to assess the performance of such kits in a polydrug context and to investigate both simple colourometric techniques and novel technologies.


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509632/1 01/10/2016 30/09/2021
2010014 Studentship EP/N509632/1 16/10/2017 30/06/2021 Andrew Brandon
Description The number of drug-related deaths in the UK continues to rise, and most involve polysubstance drug use. This study translates traditional drug discovery analytical methodologies used in the pharmaceutical industry to investigate drug metabolism, pharmacokinetics and drug-drug interactions arising from the non-medical use of traditional and novel drugs in a polysubstance misuse context. For many of these compounds, especially the newly emerging and novel drugs, little information regarding their combined biological activity and effects or their structure dependent rates of metabolic clearance is currently available. Additionally, the benzodiazepines present on the illicit (street) market can be highly variable. Multiple analytical techniques have been employed to identify and quantify the content of benzodiazepine samples seized from prisoners in Scotland, which have been anonymised and supplied by the Scottish Prison Service for non-judicial testing.

The 'conventional' prescribed and illicit drugs and many novel benzodiazepines are very metabolically stable and their intrinsic clearance and metabolite formation is very slow. It has therefore proved difficult to observe any potential changes to metabolite formation, for example, when combined in vitro using tools commonly used in the pharmaceutical industry. This has lead to an expansion of the drugs being studied to include synthetic cannabinoids which are cleared at a much faster rate than the other drugs being studied.

Intrinsic clearance rates (the base rate at which the drug is cleared by enzymes in the liver, without accounting for other factors) have been established for a wide range of drugs of interest. Structure-metabolism relationships for benzodiazepines and synthetic cannabinoids have been established. Intrinsic clearance rates of studied drugs vary widely, from the very slow (e.g. the novel benzodiazepine phenazepam) to the very fast (e.g. the synthetic cannabinoid (S)-AMB-FUBINACA). For synthetic cannabinoids, it has been observed that the conformation, chirality and the presence or absence of certain functional groups has a significant effect on the rate of their metabolism. As all of the studied synthetic cannabinoids are chiral, the research has shown that (S)- and (R)- enantiomers, both of which can theoretically be present in illicit samples, are cleared at different rates with (S)-enantiomers being cleared faster than (R)-enantiomers.

How lipophilic ('fat-loving') a drug is can heavily influence its behaviour in the body. Lipophilic drugs tend to be more potent, more easily cross into the brain to exert an effect, are more highly bound to proteins in the blood, and can reside in fat tissue for extended periods. Tested synthetic cannabinoids were found to have relatively high lipophilicity both experimentally and using computer prediction software. These compounds were also found to be stable in human plasma, demonstrating that they are not metabolised by carboxylesterase enzymes present in whole blood. Different carboxylesterases present in the liver, and to a lesser extent in some other tissues, are likely to be partly responsible for synthetic cannabinoid metabolism. The proportion of the freely circulating drug which is available to exert a pharmacological effect and to be metabolised, is dependent upon their ability to bind to proteins in the blood plasma. The protein binding of synthetic cannabinoids was tested and was found to be high, ranging from 88.9-99.5%.

Due to the high potency and illicit nature of the drugs being studied, it is not possible to study their properties in humans. It is however possible to predict the clearance rates of drugs using published prediction models and the data mentioned above. Once factors such as protein binding were accounted for, predicted clearance in humans was much slower; although synthetic cannabinoids are fundamentally metabolically unstable, the high degree of protein binding coupled with their likely accumulation in adipose tissue means that their toxicological detection windows will be extended.
Metabolising enzyme-related drug-drug interactions have been investigated for the majority of the target compounds, with some potential interactions shown to occur at high drug concentrations. For example, antidepressants duloxetine and fluoxetine, which are often prescribed to problem drug users, have shown moderate to strong inhibition of the enzymes responsible for metabolising most benzodiazepines. In addition, several synthetic cannabinoids have shown inhibition potential against a number of metabolising enzymes.

Further publications are planned, for example on the pharmacokinetics and the detection/quantification of benzodiazepines. The aim is to publish these articles around or just after the end of the funding period.
Exploitation Route By combining intrinsic clearance, protein binding and plasma stability data it has been possible to estimate human in vivo clearance rates for synthetic cannabinoids for the first time, thus adding to the fundamental bioanalytical and pharmacokinetic data available for emerging drugs (with a focus on synthetic cannabinoids and benzodiazepines). This will help toxicologists to understand the likely detection windows and harms of such substances and aid in the interpretation of toxicological data in context. The plasma protein binding of novel benzodiazepines has been studied, where data is not already available in the literature, and this will be used in predicting in vivo clearance for these compounds. Potential drug-drug interactions are being evaluated using gathered data, in the context of drug concentrations detected in post-mortem toxicology. This will be valuable in predicting the risks of combining certain drugs and may advise clinicians in selecting which drugs are appropriate for prescription to those misusing the novel substances involved in this study.

The gathering of reference data for benzodiazepines using ion mobility spectrometry will allow for those in the prison and airport sectors to successfully perform preliminary identification of these drugs in test samples, which at present cannot be done. The identification and quantification of benzodiazepines in seized samples will give an overview of the range of drugs and their concentrations present in illicit samples, so practitioners and users may be better informed on the drugs' potential content.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description The drug-related death (DRD) rate in Scotland in 2019 was among the highest reported rates in the world, and is substantially higher than both the rest of the UK and the European average. Much of the problem drug use in Scotland involves the combined use of multiple drugs (poly-drug misuse), mostly central nervous system depressants. When certain drugs are combined, for example opioids, benzodiazepines and/or gabapentinoids, the risk of overdose increases significantly. The fundamental physicochemical properties and pharmacokinetics of commonly misused drugs have been investigated, providing protocols for the study of emerging drug threats, with interpretation of potential interactions in a polysubstance misuse context. This will help in developing risk assessments related to the combinational use of both prescription and illicitly produced drugs. The drugs being studied include commonly misused opioids, anticonvulsants, antidepressants and a range of benzodiazepines. Additionally, a wide range of synthetic cannabinoids whose use is highly prevalent within the prison population are being studied. Among problem drug users in Scotland, heavy use of benzodiazepines is common. Since the rise of new/novel psychoactive substances in recent years, these street benzodiazepine tablets can now contain any of a wide range of benzodiazepine-type compounds, for many of which little information is known. It is important that the pharmacokinetics of these drugs are better understood as to increase knowledge on their metabolism and effects in the body, such as how long the parent compounds last and where in the body they are broken down. This will improve understanding of their harms and help clinicians in deciding which body fluids to test for these drugs as well as how long the detection windows for parent compounds are. Reference data for the identification of benzodiazepines using ion mobility spectrometry may be used in both prisons and airports, while quantification of seized benzodiazepines will improve understanding of the range in dosage they contain. Within prisons in Scotland, in addition to the above mixtures, there is also widespread heavy use of synthetic cannabinoids. For these compounds, next to no information on their pharmacokinetics was previously known. The collection of novel information on these provides a fundamental understanding of the stability and potential interactive effects of these compounds within the body. This basic information has provided a better understanding of their chemistry and their risks to the users and those around them. Through assessment of cytochrome P450 enzyme inhibition, in combination with other gathered data, it may also be possible to advise on the potential risks and interactions with specific combinations. Prescribers once aware of this information could make informed decisions on which drugs are appropriate to prescribe to individuals who are or may be misusing the drugs involved in this study.
First Year Of Impact 2019
Impact Types Societal

Title A Systematic Study of the In Vitro Pharmacokinetics and Estimated Human In Vivo Clearance of Indole and Indazole-3-Carboxamide Synthetic Cannabinoid Receptor Agonists Detected on the Illicit Drug Market 
Description Raw data as supplementary information to the publication "A Systematic Study of the In Vitro Pharmacokinetics and Estimated Human In Vivo Clearance of Indole and Indazole-3-Carboxamide Synthetic Cannabinoid Receptor Agonists Detected on the Illicit Drug Market", available at the following link: 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Publication of this dataset allows transparency of data and provides raw data for the publication of the same title. This will allow fellow researchers to better understand the pharmacokinetic properties of these drugs as well as any variation within or between compounds. Availability of this data will also aid in the conducting of similar studies or in repeating this work. 
Description Division of Drug Research, Linköping University 
Organisation Linkoping University
Country Sweden 
Sector Academic/University 
PI Contribution The supplied synthetic cannabinoid metabolites will be used in studying their pharmacokinetics. This will include study of their intrinsic clearance rates, plasma stability, plasma protein binding and most likely isozyme mapping - identification of the enzymes responsible for the further breakdown of these metabolites. These metabolites are longer lasting in the body than the parent compounds and may also have pharmacological effects themselves. Thus it is important that data is gathered on these metabolites as these are likely to be used as markers in toxicology casework for consumption of the parent drug.
Collaborator Contribution This collaboration is with Prof. Henrik Green and Dr. Shimpei Watanabe (Division of Drug Research, Department of Biomedical and Clinical Sciences, Linköping University, Sweden). They have kindly synthesised six metabolites of two currently circulating synthetic cannabinoids (three for each) and supplied these free of charge. Studies on these compounds performed by the collaborators will be combined with those done in this project in at least one scientific paper.
Impact Metabolites have only recently been supplied, and in vitro work on these compounds is currently underway.
Start Year 2019
Description PhD supervisory team 
Organisation University of Glasgow
Department College of Medical, Veterinary and Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This project is being conducted in collaboration with Denise McKeown (Forensic Medicine and Science, University of Glasgow), a senior toxicologist who is acting as an external co-supervisor on this project. The outcome of this project will provide up to date information on the pharmacokinetics of numerous novel and 'conventional' drugs which will of great value to forensic toxicologists and clinicians. As a working toxicology laboratory, this group will benefit from the information gathered in this project.
Collaborator Contribution This project is being conducted in collaboration with Denise McKeown (Forensic Medicine and Science, University of Glasgow), a senior toxicologist who is acting as an external co-supervisor on this project. Her role is to provide advice and guidance to ensure the project is relevant to and addresses issues encountered by toxicologists and clinicians in the field.
Impact This partnership, with Denise acting as external co-supervisor ensures that the project remains relevant to and addresses issues encountered by toxicologists and clinicians in the field. Thus the outcomes of this project will be of great use to toxicologists, prescribers and the like.
Start Year 2017
Description Oral presentation - Scottish Forensic Research Student Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This is a symposium for forensic research students throughout Scotland, as well as some researchers and industry members. I gave an oral presentation on my research to the audience which was well received and prompted several questions from the audience. The presentation was awarded sponsor's choice for which I received a certificate and small prize.
Year(s) Of Engagement Activity 2019
Description Poster presentation - Scottish Forensic Research Student Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This was a yearly symposium/conference targeted towards forensic research students. This allowed me to convey my research to a range of students and researchers who study forensics. Two awards were given for the poster - one from each sponsor for sponsor's choice of best poster.
Year(s) Of Engagement Activity 2018
Description Poster presentation - The International Association of Forensic Toxicologists (TIAFT) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Aspects of this work was presented as a poster at The International Association of Forensic Toxicologists (TIAFT) meeting held in Ghent, Belgium, August 2018. This is an international conference for forensic toxicologists and researchers, with around 800 attendees.
Year(s) Of Engagement Activity 2018
Description Presentations within the University of Dundee 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Several presentations of this work have been given within the University of Dundee, delivered to a diverse range of staff members and postgraduate research students.
Year(s) Of Engagement Activity 2018,2019,2020
Description Three Minute Thesis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The 'Three-minute thesis' competition was entered, which involved presenting this work in an oral presentation of 3 minutes or less to a public audience, with a single static slide. This is an international competition. There was a high turnout with regard to the audience.
Year(s) Of Engagement Activity 2019