Cannabis sativa L., a plant known for a long time for its pharmacological properties, is currently approved for various therapeutic indications, such as the treatment of muscular and neuropathic pain, epilepsy, and multiple sclerosis. However, still very little is known about the complex chemical composition of cannabis extracts. Over 150 active terpenophenolic components called phytocannabinoids are known to be present in the plant, but only a few of them have been isolated and characterized. It is important to characterize the phytocannabinoid composition of cannabis extracts as it influences the pharmacological profile. In light of this, my doctoral project aims to identify and quantify new phytocannabinoids, and evaluate their pharmacological activity. In the first part of my PhD program, an analytical method based on high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was developed in an untargeted metabolomics fashion. This allowed for the putative identification of numerous carboxylated and decarboxylated phytocannabinoids, including Cannabidihexol (CBDH), Tetrahydrocannabihexol (Δ9-THCH), Cannabigerobutol (CBGB), Cis-Δ9-tetrahydrocannabinolic acid (cis- Δ9-THCA), Δ9-tetrahydrocannabiphorolic acid (Δ9-THCPA), Cannabidiphorolic acid (CBDPA). To confirm their identity, these compounds were isolated, characterized, and their chemical properties were compared to those of the corresponding synthetic species obtained through stereoselective synthesis developed ad hoc. The latter were used as reference standards for the development of sensitive and selective HPLC-UV-HRMS methods under a targeted metabolomics fashion in order to quantify the new phytocannabinoids in different cannabis varieties. Such new compounds were further subjected to in vitro and in vivo tests to assess their pharmacological profile. In the second part, the stereoisomeric composition of the two major phytocannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), as well as of their carboxylated precursors, Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and cannabidiolic acid (CBDA), was investigated in the Italian medicinal cannabis variety FM2. Bidimensional achiral-chiral HPLC methods coupled to UV and HRMS were developed and optimized for this purpose. Ultimately, the project focused on the study of new psychoactive substances (NPS), psychoactive drugs not controlled by the Single Convention on Narcotic Drugs (1961) or the Convention on Psychotropic Substances (1971), but which may pose a public health threat. Specifically, two NPS derived from CBD, a non-psychoactive component of cannabis, were investigated: hexahydrocannabinol (HHC) and tetrahydrocannabinol (H4-CBD). Synthetic strategies were developed to obtain epimers of both HHC and H4-CBD which were used as reference standards for the development of quantitative analytical methods to be applied to commercial samples. For HHC epimers, an HPLC-HRMS method was developed, while for H4-CBD epimers, known to have cannabinomimetic activity, a qualitative-quantitative gas chromatography-mass spectrometry (GC-MS) method was developed. Furthermore, HHC epimers were purified and individually tested for their cannabinomimetic activity. As a result of the present PhD study, new cannabinoids have been added to the inventory, providing an increasingly comprehensive overview of the phytocannabinome. The project also supplied sensitive and specific HPLC-UV-HRMS methods for the qualitative-quantitative and stereoisomeric evaluation of phytocannabinoids in Cannabis sativa L. extracts. Additionally, HPLC-HRMS and GC-MS methods were provided for the qualitative and quantitative analysis of semisynthetic CBD derivatives in real samples.
La Cannabis sativa L., pianta conosciuta da tempo per le sue proprietà farmacologiche, è attualmente approvata per diverse applicazioni terapeutiche come il trattamento del dolore muscolare e neuropatico, epilessia e sclerosi multipla. Tuttavia, si sa molto poco sulla complessa composizione chimica dell’estratto di cannabis. Oltre 150 diversi componenti attivi a struttura terpenofenolica, chiamati fitocannabinoidi, sono noti nella pianta, ma solo pochi di essi sono stati isolati e caratterizzati. È importante caratterizzare la composizione in fitocannabinoidi negli estratti in quanto essa influenza l’effetto farmacologico sul paziente. Alla luce di ciò, il mio progetto di dottorato ha lo scopo di identificare e quantificare nuovi fitocannabinoidi per poi valutarne l’attività farmacologica. Nella prima fase del progetto di dottorato, ho sviluppato un metodo di cromatografia liquida accoppiata a spettrometria di massa ad alta risoluzione (HPLC-HRMS) con approccio metabolomico untargeted. Ciò ha permesso di identificare putativamente numerosi fitocannabinoidi carbossilati e decarbossilati come: Cannabidiexolo, Tetraidrocannabiexolo, Cannabigerobutolo, acido cis-Δ9-tetraidrocannabinolico, acido Δ9-tetraidrocannabiforolico, acido cannabidiforolico. Per confermarne l’identità sono stati isolati dall’estratto e caratterizzati e le loro proprietà chimiche sono state confrontate con quelle degli analoghi sintetici ottenuti mediante sintesi stereoselettive sviluppate ad hoc. Questi ultimi sono stati utilizzati come standard nello sviluppo di metodi HPLC-HRMS basati su un approccio metabolomico targeted al fine di quantificare i nuovi fitocannabinoidi in diverse varietà di cannabis. Inoltre, tali nuovi composti sono stati impiegati in test in vitro e in vivo per studiarne il profilo farmacologico. Nella seconda fase del progetto di dottorato, ho indagato il comportamento stereochimico dei due principali fitocannabinoidi, Δ9-tetraidrocannabinolo (Δ9-THC) e cannabidiolo (CBD), nonchè dei rispettivi precursori carbossilati, acido Δ9-tetrahydrocannabinolico (Δ9-THCA) e acido cannabidiolico (CBDA) nell’estratto di cannabis medicinale italiana FM2. A tale scopo sono stati sviluppati e ottimizzati metodi HPLC bidimensionale achirale-chirale accoppiati ad UV e HRMS. In ultima analisi, il progetto ha riguardato lo studio di nuove sostanze psicoattive (NPS), nuove droghe psicotrope non controllate dalla Single Convention on Narcotic Drugs (1961) o dalla Convention on Psychotropic Substances (1971), ma che possono rappresentare una minaccia per la salute pubblica. In particolare, sono stati investigati due NPS derivanti dal CBD, componente non psicoattivo della cannabis: esaidrocannabinolo (HHC) e tetraidrocannabidiolo (H4-CBD). Sono quindi state messe a punto strategie sintetiche per ottenere gli epimeri di entrambi i derivati, i quali sono stati poi impiegati come standard per lo sviluppo di metodi analitici quantitativi da applicare a campioni commerciali. Per l’HHC è stato sviluppato un metodo HPLC-HRMS, mentre per gli epimeri dell’H4-CBD è stato messo a punto un metodo basato su gas cromatografia accoppiata a spettrometria di massa (GC-MS). Inoltre, i due epimeri dell’HHC sono stati purificati e testati singolarmente per la loro attività cannabinomimetica. Attraverso tale studio nuovi cannabinoidi sono stati aggiunti all’inventario fornendo una panoramica sempre più completa del fitocannabinoma. Inoltre, questo lavoro di tesi ha fornito nuovi metodi HPLC-UV-HRMS sensibili e specifici per la valutazione della composizione quali-quantitativa e stereoisomerica di fitocannabinoidi in estratti di C. sativa L. Questo studio ha infine fornito metodi HPLC-HRMS e GC-MS per analisi quali-quantitative di derivati semisintetici del CBD in campioni reali.
Sviluppo di strategie sintetiche e di metodi analitici basati su cromatografia liquida accoppiata a spettrometria di massa ad alta risoluzione (HPLC-HRMS) per la caratterizzazione chimico-farmaceutica di principi attivi degli estratti di Cannabis Sativa L. e di nuove sostanze psicotrope (NPS)
RUSSO, FABIANA
2024
Abstract
Cannabis sativa L., a plant known for a long time for its pharmacological properties, is currently approved for various therapeutic indications, such as the treatment of muscular and neuropathic pain, epilepsy, and multiple sclerosis. However, still very little is known about the complex chemical composition of cannabis extracts. Over 150 active terpenophenolic components called phytocannabinoids are known to be present in the plant, but only a few of them have been isolated and characterized. It is important to characterize the phytocannabinoid composition of cannabis extracts as it influences the pharmacological profile. In light of this, my doctoral project aims to identify and quantify new phytocannabinoids, and evaluate their pharmacological activity. In the first part of my PhD program, an analytical method based on high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was developed in an untargeted metabolomics fashion. This allowed for the putative identification of numerous carboxylated and decarboxylated phytocannabinoids, including Cannabidihexol (CBDH), Tetrahydrocannabihexol (Δ9-THCH), Cannabigerobutol (CBGB), Cis-Δ9-tetrahydrocannabinolic acid (cis- Δ9-THCA), Δ9-tetrahydrocannabiphorolic acid (Δ9-THCPA), Cannabidiphorolic acid (CBDPA). To confirm their identity, these compounds were isolated, characterized, and their chemical properties were compared to those of the corresponding synthetic species obtained through stereoselective synthesis developed ad hoc. The latter were used as reference standards for the development of sensitive and selective HPLC-UV-HRMS methods under a targeted metabolomics fashion in order to quantify the new phytocannabinoids in different cannabis varieties. Such new compounds were further subjected to in vitro and in vivo tests to assess their pharmacological profile. In the second part, the stereoisomeric composition of the two major phytocannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), as well as of their carboxylated precursors, Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and cannabidiolic acid (CBDA), was investigated in the Italian medicinal cannabis variety FM2. Bidimensional achiral-chiral HPLC methods coupled to UV and HRMS were developed and optimized for this purpose. Ultimately, the project focused on the study of new psychoactive substances (NPS), psychoactive drugs not controlled by the Single Convention on Narcotic Drugs (1961) or the Convention on Psychotropic Substances (1971), but which may pose a public health threat. Specifically, two NPS derived from CBD, a non-psychoactive component of cannabis, were investigated: hexahydrocannabinol (HHC) and tetrahydrocannabinol (H4-CBD). Synthetic strategies were developed to obtain epimers of both HHC and H4-CBD which were used as reference standards for the development of quantitative analytical methods to be applied to commercial samples. For HHC epimers, an HPLC-HRMS method was developed, while for H4-CBD epimers, known to have cannabinomimetic activity, a qualitative-quantitative gas chromatography-mass spectrometry (GC-MS) method was developed. Furthermore, HHC epimers were purified and individually tested for their cannabinomimetic activity. As a result of the present PhD study, new cannabinoids have been added to the inventory, providing an increasingly comprehensive overview of the phytocannabinome. The project also supplied sensitive and specific HPLC-UV-HRMS methods for the qualitative-quantitative and stereoisomeric evaluation of phytocannabinoids in Cannabis sativa L. extracts. Additionally, HPLC-HRMS and GC-MS methods were provided for the qualitative and quantitative analysis of semisynthetic CBD derivatives in real samples.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/157043
URN:NBN:IT:UNIMORE-157043