Medicinal cannabis: Principal cannabinoids concentration and their stability evaluated by a high performance liquid chromatography coupled to diode array and quadrupole time of flight mass spectrometry method.

In the last few years, there has been a boost in the use of cannabis-based extracts for medicinal purposes, although their preparation procedure has not been standardized but rather decided by the individual pharmacists. The present work describes the development of a simple and rapid high performance liquid chromatography method with UV detection (HPLC-UV) for the qualitative and quantitative determination of the principal cannabinoids (CBD-A, CBD, CBN, THC and THC-A) that could be applied to all cannabis-based medicinal extracts (CMEs) and easily performed by a pharmacist. In order to evaluate the identity and purity of the analytes, a high-resolution mass spectrometry (HPLC-ESI-QTOF) analysis was also carried out. Full method validation has been performed in terms of specificity, selectivity, linearity, recovery, dilution integrity and thermal stability. Moreover, the influence of the solvent (ethyl alcohol and olive oil) was evaluated on cannabinoids degradation rate. An alternative extraction method has then been proposed in order to preserve cannabis monoterpene component in final CMEs.

"Heart-cut" bidimensional achiral-chiral liquid chromatography applied to the evaluation of stereoselective metabolism, in vivo biological activity and brain response to chiral drug candidates targeting the central nervous system.

A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC-LC-MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the "heart-cut" two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal.

7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-Dioxide as Positive Allosteric Modulator of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor. The End of the Unsaturated-Inactive Paradigm?

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1.

Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide.

Chiral 5-arylbenzothiadiazine derivatives have recently attracted particular attention because they exhibit an interesting pharmacological activity as AMPA receptor (AMPAr) positive modulators. However, investigations on their configurational stability suggest a rapid enantiomerization in physiological conditions. In order to enhance configurational stability, preserving AMPAr activity, we have designed the novel compound (R,S)-7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzothiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute configuration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-flow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound.

Development of an in vitro liquid chromatography-mass spectrometry method to evaluate stereo and chemical stability of new drug candidates employing immobilized artificial membrane column.

A stopped-flow HPLC method was developed to evaluate configurational and chemical stability of pharmaceutical compounds employing immobilized artificial membranes (IAM) column to simulate conditions that pharmaceutical compounds will meet in vivo. The method was applied to recent developed chiral 5-arylbenzothiadiazine derivatives possessing high positive allosteric modulatory (PAM) activity on AMPA receptor. In particular the stopped-flow HPLC method developed used a chiral column to separate single enantiomer of the compounds that are forced into an IAM column where configurational and chemical stability was evaluated in simulated gastrointestinal fluids (pH 1.2 and 6.8 at 37.5 °C) to simulate in vivo conditions. The results were compared to those obtained by dynamic and off-column methods to evaluate the effects of stationary phases on kinetic constant of enantiomerization and hydrolysis. The results suggested that the phospholipids environment of IAM stationary phases, which mimes biological membrane, greatly influence the hydrolysis process increasing the chemical stability of tested compounds while no influence on enantiomerization kinetic was observed. Therefore it is possible to suppose that 5-arylbenzothiadiazine derivatives should not hydrolysed in vivo while they should rapidly racemized in aqueous solvents. The method could represents a rapid and value tool to predict chemical and configurational stability of new chemical entities to decrease the number of animal studies.

Simultaneous measurement of adenosine, dopamine, acetylcholine and 5-hydroxytryptamine in cerebral mice microdialysis samples by LC-ESI-MS/MS.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous measurement of adenosine (ADE), dopamine (DA), acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) in mouse brain microdialysates. High method sensitivity (LLOQ of 0.05nM) was achieved by optimization of chromatographic and mass spectrometric parameters. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. The LC-MS/MS method was successfully applied to evaluate the effect of the systemic administration of cocaine or amphetamine on the extracellular levels of ADE, DA, ACh and 5-HT in the mouse nucleus accumbens by microdialysis.

An improved LC-S/MS method for the quantitation of adenosine concentration in mice brain microdialysates.

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of adenosine concentrations in mouse brain microdialysis samples was developed. High method sensitivity (LLOQ of 1.25 fmol) was achieved by on-line switching column. A C18 was employed as enrichment column and a cyano based (CN-SB) as analytical column. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. It was successfully applied to measure quantitatively adenosine in brain of freely moving mice after different stimuli.

5-Arylbenzothiadiazine Type Compounds as Positive Allosteric Modulators of AMPA/Kainate Receptors.

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPAr) has led to the search for new AMPAr positive modulators. On the basis of crystallographic data of the benzothiadiazines binding mode in the S1S2 GluA2 dimer interface, a set of 5-aryl-2,3-dihydrobenzothiadiazine type compounds has been synthesized and tested. Electrophysiological results suggested that 5-heteroaryl substituents on the benzothiadiazine core like 3-furanyl and 3-thiophenyl dramatically enhance the activity as positive modulators of AMPAr with respect to IDRA21 and cyclothiazide. Mouse brain microdialysis studies have suggested that 7-chloro-5-(3-furyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide crosses the blood-brain barrier after intraperitoneal injection. Biological results have been rationalized by a computational docking simulation that it has currently employed to design new AMPAr-positive modulator candidates.

Molecular modeling studies, synthesis, configurational stability and biological activity of 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide.

The potential therapeutic benefit of compounds able to activate AMPA receptors (AMPArs) has led to a search for new AMPAr positive modulators. Among them, 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide (1) has attracted particular attention, because it is one of the most active benzothiadiazine-derived positive modulators of the AMPA receptor. It possesses two stereogenic centers, C3 and C6, thus it can exist as four stereoisomers. In this work, preliminary in silico studies suggested that 1 interacts stereoselectively with AMPArs. Single stereoisomers of 1 were prepared in order to evaluate their biological activity. However, studies regarding the configurational stability of the investigated compounds suggested a rapid epimerization at C3 in aqueous solvents, and we can expect the same reaction in vivo. Thus, electrophysiological experiments were performed on the two epimeric mixtures, (3∗,6R)- and (3∗,6S)- 8-chloro-2,3,5,6-tetrahydro-3,6-dimethyl-pyrrolo[1,2,3-de]-1,2,4-benzothiadiazine 1,1-dioxide, in order to evaluate their activities as positive allosteric modulators of AMPArs. The obtained data suggest that the (3∗,6S) epimeric mixture is the most active in positively modulating AMPArs, confirming in silico results.

Evaluation of stereo and chemical stability of chiral compounds.

A stopped-flow bidimensional recycle HPLC (sf-BD-rHPLC) configuration has been used to investigate simultaneously the stereo and chemical stability of labile chiral compounds. The single enantiomers of a racemate can be separated on chiral column (first dimension) and each one can be trapped in the achiral column (second dimension) that works as reactor.By filling the achiral column with the appropriate aqueous buffers it is possible to evaluate the stability of the trapped enantiomer toward aqueous buffer itself. It was possible to recycle the reaction products formed in the chiral column (first dimension) where they are separated by a second six valve port. The reaction rate constants were calculated for the different processes occurred in the achiral column by means of corresponding peak areas. The method was applied to a pharmacological active compound: (±)7-chloro-5-ethyl-3-methyl-3,4-dihydro-2H-benzo[1,2,4]thiadiazine 1,1-dioxide ((±)-1) to evaluate enantiostability and hydrolysis in conditions similar to those of biological fluid. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis and enantiomerization at the same temperature and in the same solvents used in sf-BD-rHPLC. The good agreement of the results obtained validate the novel procedure developed. Furthermore, the results generated off-line were used to determine the influence of solvents on the racemization of (±)-1.

Epimerization and hydrolysis of 3,6-dimethyl-2,3,5,6-tetrahydro[1,2,4]thiadiazino[6,5,4-hi]indole 1,1-dioxide.

In this study, configurational and chemical stability of (R,R),(S,S),(R,S),(S,R)-3,6-dimethyl-2,3,5,6-tetrahydro[1,2,4]thiadiazino[6,5,4-hi]indole 1,1-dioxide (1) were investigated by dynamic and stopped-flow HPLC methods. Single epimeric mixtures (R,R),(R,S)-1 and (S,S),(S,R)-1 were obtained combining synthetic and chromatographic strategies. Separation of (R,R)-1 and (R,S)-1 was achieved by chiral chromatography and absolute configuration of eluted epimers has been assigned basing on molecular modelling calculations. Epimerization and hydrolysis of (R,R),(R,S)-1 have been studied by classical off-column, dynamic HPLC and stopped-flow HPLC methods. The influence of different parameters, such as temperature, pH and dielectric constant was evaluated. The data obtained indicate that (R,R),(R,S)-1 undergoes to a rapid epimerization in aqueous solvent and hydrolysis in acidic conditions. Moreover, epimerization and hydrolysis were investigated in presence of an artificial membrane and in physiological buffers (pH 2.2 and 7.0 at 37.5°C) to simulate in vivo conditions.

Quantitative analysis of acetylcholine in rat brain microdialysates by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the quantitative analysis of acetylcholine in rat brain dialysates. The separation of acetylcholine (ACh), choline (Ch), acetyl-ß-methylcholine (IS) from endogenous compounds and Ringer's salts was achieved with cation exchange chromatography. Optimization of chromatographic and mass spectrometry parameters were perfomed in order to improve sensitivity of the method. The limit of detection were 0.05 and 3.75 fmol on column with S/N ratio of 3:1 for ACh and Ch, respectively. The limit of quantitation (LOQ) for ACh and Ch measured in Ringer's solution were 0.05 nM (0.25 fmol) and 3.75 nM (18.75 fmol), respectively at S/N ratio of 10:1. Linearity of the method has been evaluated in the concentrations range between 0.05 and 5.00 nM and 3.75 and 200 nM for ACh and Ch respectively. The correlation coefficients were 0.999 and 0.995 for ACh and Ch respectively, indicating very good linearity. The LC/MS/MS method developed has been applied to evaluate the effect of oral administration of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21), a positive modulators of AMPA receptor, on the release of ACh in the rat prefrontal cortex by microdialysis.

Determination of kinetic parameters of enantiomerization of benzothiadiazines by DCXplorer.

Benzothiadiazines differently substituted at the sulfonamidic nitrogen atom, at the stereogenic carbon atom and at the anilinic nitrogen atom have been synthesized and fully characterized. Enantioseparation of these compounds has revealed rapid on-column enantiomerization. The recently developed software DCXplorer has been successfully applied to calculate enantiomerization kinetic parameters. Enantiomerization barriers of 3-phenyl substituted benzothiadiazines, calculated in this work, have indicated a higher enantiomerization rate suggesting that the aromatic substituent exerts a strong effect on the enantiomerization process. Methyl substitution on N(2) position led to higher free energy barriers of enantiomerization, suggesting negative influence of methyl in the N(2) position on enantiomerization kinetics. However, methylation on N(4) position increases the enantiomerization rates significantly. The results obtained have been employed to postulate an enantiomerization mechanism for chiral benzothiadiazine type compounds.

Simultaneous determination of enantiomerization and hydrolysis kinetic parameters of chiral N-alkylbenzothiadiazine derivatives.

On-column stopped flow multidimensional HPLC (sfMDHPLC) and dynamic high-performance liquid chromatography were applied to investigate the influence of alkyl substituents at the sulfonamidic and amino moieties of benzothiadiazine 1,1-dioxide derivatives on hydrolysis and enantiomerization rate constants. The data obtained indicate the presence of pyrrolo substituent at the 3,4 positions on benzothiadiazine rings inhibits the hydrolysis, whereas the enantiomerization occurs in acidic medium. Hydrolysis rates are quite similar for the two benzothiadiazines methyl substituted to nitrogen at 2- and 4-positions. Conversely, enantiomerization rate of 4-N-methyl substituted is significantly higher than 2-N-methyl substituted.

On-line racemization by high-performance liquid chromatography.

An on-column stopped-flow bidimensional recycling HPLC procedure was developed to obtain an enantiomeric enrichment starting from a racemic mixture. The method developed was applied to two chiral compounds of pharmaceutical interest, (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide (1) and (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-)IDRA21, (2)), since the pharmacological activity of the two benzothiadiazine derivatives investigated has been ascribed to only one enantiomer. Starting from a racemic mixture it was possible to obtain about 95% of pure enantiomer. The procedure was applied both in reverse-phase mode and in normal-phase mode. The scaled up and automatization of the novel analytical HPLC procedure represents a powerful tool to obtain pure enantiomer starting from racemic compounds without cumbersome stereoselective synthesis or expensive enantiopurification processes.

A novel class of allosteric modulators of AMPA/Kainate receptors.

The rapid hydrolysis in vivo of IDRA21 to 2-amino-5-chlorobenzensulfonamide has been demonstrated by microdialysis experiments. The IDRA21 metabolite possess in vitro a biological activity similar to that of IDRA21 itself. Taking 2-amino-5-chlorobenzensulfonamide as lead compound, a novel class of AMPAR positive allosteric modulators has been prepared.

Enantiomerization and hydrolysis of (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide by stopped-flow multidimensional high-performance liquid chromatography.

A novel stopped-flow multidimensional HPLC (sf-MD-HPLC) procedure has been developed to investigate simultaneously the effect of the pH on the enantiostability and hydrolysis of (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide [(+/-)IDRA21]. It was possible to determinate the rate constants and free energy barriers of enantiomerization and hydrolysis rate constants of (+/-)IDRA21, by using two chiral stationary phases (CSPs) and one achiral C18 column. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis at the same temperature and in the same buffers used in sf-MD-HPLC. The good agreement of the results obtained validate the sf-MD-HPLC procedure. Furthermore, hydrolysis rate constants of (+/-)IDRA21 were calculated in a series of buffers over a pH range of 1.20-10.60 at 37 degrees C in order to evaluate the influence of the pH on hydrolysis.

Enantioseparation of the antidepressant reboxetine.

The enantioseparation of reboxetine by HPLC was investigated using chiral stationary phases (CSPs) containing cellulose Tris(3,5-dimethylphenyl)carbamate on silica gel (Chiralcel OD column) as the chiral selector. Reversed phase HPLC was the technique of choice for the analytical enantioseparation of reboxetine, while the chiral semipreparative separation was obtained with the same CSP, but in normal phase conditions. The effects of the mobile phase pH and composition on analytical retention, enantioselectivity and resolution were investigated. The best performance was obtained using a mobile phase composed of 0.5M sodium perchlorate at pH 6 and acetonitrile in the 60/40 (v/v) ratio. The semipreparative separation has allowed obtaining pure enantiomers, but required the preparation of reboxetine free base. Different n-hexane/alcohol mixtures were tested as mobile phases, varying both the nature of the alcohol and its percentage in the mobile phase. Different n-hexane/alcohol mixtures were tested as mobile phase and the best results were obtained by using a mobile phase composed of n-hexane and 2-propanol (80:20, v/v).

Enantiomerization of chiral 2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4] benzothiadiazine 5,5-dioxide by stopped-flow multidimensional HPLC.

An on-column stopped-flow multidimensional HPLC (sfMDHPLC) procedure using two chiral stationary phases (CSPs) and one achiral C18 column was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide. Moreover, a stopped-flow HPLC (sfHPLC) method previously developed was applied to the determination of kinetic parameters of enantiomerization of the above compound in the presence of a CSP. The individual enantiomers of the studied compound were isolated in parallel by preparative HPLC and the rate constants and free energy barriers of enantiomerization were determined in different solvents (off-column method). The data obtained by sfMDHPLC, sfHPLC and off-column methods were compared. The (S) enantiomer of the studied compound (S18986) was prepared by asymmetric synthesis and subsequently purified by preparative HPLC, followed by the determination of rate constants and free energy barriers of enantiomerization in different buffer solutions at pH 2-9.3.

1,2,4-Benzothiadiazine derivatives as alpha1 and 5-HT1A receptor ligands.

A series of new 1,2,4-benzothiadiazine derivatives with an arylpiperazine mojety linked at position 3 of the heterocyclic ring were synthesized and assessed for their pharmacological profiles at alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) by functional experiments and by in vitro binding studies at human cloned 5-HT(1A) receptor. Compound 1 was identified as a novel alpha(1D) antagonist (pK(b)alpha(1D)=7.59; alpha(1D)/alpha(1A)>389; alpha(1D)/alpha(1B)=135) with high selectivity over 5-HT(1A) receptor (5-HT(1A)/alpha(1D)<0.01), while compound 6, a 3,4-dihydro-derivative, was characterized as a novel 5-HT(1A) receptor ligand, highly selective over alpha(1D)-adrenoceptor subtype (pK(i)5-HT(1A)=8.04; 5-HT(1A)/alpha(1D)=1096). Further pharmacological studies demonstrated that 6 is a partial agonist at 5-HT(1A) receptor (E(max)=23, pD(2)=6.92).