| 1. |
- Larsson, Johan, 1990-, et al.
(författare)
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Combining endocannabinoids with retigabine for enhanced M-channel effect and improved KV7 subtype selectivity
- 2020
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Ingår i: The Journal of General Physiology. - : ROCKEFELLER UNIV PRESS. - 0022-1295 .- 1540-7748. ; 152:8
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Tidskriftsartikel (refereegranskat)abstract
- Retigabine is unique among anticonvulsant drugs by targeting the neuronal M-channel, which is composed of KV7.2/KV7.3 and contributes to the negative neuronal resting membrane potential. Unfortunately, retigabine causes adverse effects, which limits its clinical use. Adverse effects may be reduced by developing M-channel activators with improved KV7 subtype selectivity. The aim of this study was to evaluate the prospect of endocannabinoids as M-channel activators, either in isolation or combined with retigabine. Human KV7 channels were expressed in Xenopus laevis oocytes. The effect of extracellular application of compounds with different properties was studied using two-electrode voltage clamp electrophysiology. Site-directed mutagenesis was used to construct channels with mutated residues to aid in the mechanistic understanding of these effects. We find that arachidonoyl-L-serine (ARA-S), a weak endocannabinoid, potently activates the human M-channel expressed in Xenopus oocytes. Importantly, we show that ARA-S activates the M-channel via a different mechanism and displays a different KV7 subtype selectivity compared with retigabine. We demonstrate that coapplication of ARA-S and retigabine at low concentrations retains the effect on the M-channel while limiting effects on other KV7 subtypes. Our findings suggest that improved KV7 subtype selectivity of M-channel activators can be achieved through strategically combining compounds with different subtype selectivity.
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| 2. |
- Wallgren, Jakob, 1987-
(författare)
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An insight into the metabolism of New Psychoactive Substances : Structural elucidation of urinary metabolites of synthetic cannabinoids and fentanyl analogues using synthesized reference standards
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- New Psychoactive Substances (NPS) is an umbrella term covering hundreds of substances across different drug groups. Many of these substances were originally developed for therapeutic use but have later appeared on the recreational drug market. The use of NPS has been associated with many outbreaks leading to hospitalizations and has been implicated in numerous fatalities worldwide. To be able to analytically detect drugs in a forensic setting is vital in the fight against the abuse of NPS. One of the most notable challenges in detection of NPS is the identification of major urinary metabolites for use as biomarkers. Furthermore, given the lack of reference standards in most metabolism studies, the major urinary metabolites can often only be tentatively determined.This thesis describes the synthesis and analysis of potential metabolites used to identify the exact structures of major metabolites of the synthetic cannabinoid AKB-48, fentanyl and five fentanyl analogues in authentic human urine samples and/or hepatocyte incubations. Synthetic targets were chosen based on previous metabolism studies by our research group. Subsequently, synthetic routes were developed to produce numerous potential metabolites across the studied NPS. The synthesized reference standards were analyzed by LC-QTOF-MS alongside hepatocyte drug incubations and authentic human urine samples. Comparison of the resulting analytical data was used to determine the exact structures of many metabolites. This includes urinary metabolites of AKB-48 with a single hydroxyl group situated on a secondary carbon of the adamantane moiety, or position 3 or 5 of the pentyl side chain. For the studied fentanyls, the β-OH and the 4’-OH metabolites were abundant metabolites identified in hepatocyte incubations while the 4’-OH, 4’-OH-3’-OMe and 3’,4’-diOH were the favored metabolic motifs among the metabolites identified in urine.Additionally, a concise synthetic route to produce synthetic cannabinoid metabolites with the 4-OH-5F pentyl side chain motif was developed and demonstrated for four synthetic cannabinoids. These findings and the developed synthetic routes can be used to provide forensic toxicology laboratories with urinary biomarkers for drug detection. Moreover, the synthesized reference standards of major metabolites can be studied to better understand the toxicity of their parent drugs.
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| 3. |
- Wallgren, Jakob, 1987-, et al.
(författare)
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Structure elucidation of urinary metabolites of fentanyl and five fentanyl analogues using LC-QTOF-MS, hepatocyte incubations and synthesized reference standards
- 2020
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Ingår i: Journal of Analytical Toxicology. - : Oxford University Press. - 0146-4760 .- 1945-2403. ; 44:9
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Tidskriftsartikel (refereegranskat)abstract
- Fentanyl analogues constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism and studies utilizing LC-QTOF-MS analysis of hepatocyte incubations and/or authentic urine samples does not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study seven motifs (2-, 3-, 4- and β-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogues, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC-QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogues, β-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. β-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/β-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogues where nothing is known about the metabolism.
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