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Mar 26, 2017 - ... Method for the Quantification of Cannabidiol, Cannabidivarin, Δ9-Tetrahydrocannabivarin, and Cannabigerol in Mouse Peripheral Tiss...
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Development of a rapid LC-MS/MS method for the quantification of cannabidiol, cannabidivarin, #-tetrahydrocannabivarin and cannabigerol in mouse peripheral tissues. 9

Fabiana Piscitelli, Ester Pagano, Anna Lauritano, Angelo A. Izzo, and Vincenzo Di Marzo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01094 • Publication Date (Web): 26 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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Development of a rapid LC-MS/MS method for the quantification of cannabidiol, cannabidivarin, ∆9-tetrahydrocannabivarin and cannabigerol in mouse peripheral tissues. Fabiana Piscitelli1,3*, Ester Pagano2,3, Anna Lauritano1, Angelo A. Izzo2,3, Vincenzo Di Marzo1,3*. (1) Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples. (2) Department of Pharmacy, University of Naples Federico II, Naples, Italy. (3) Endocannabinoid Research Group (ERG) Any correspondence should be addressed to these authors at: Professor Vincenzo Di Marzo, Endocannabinoid Research Group, Institute of Biomolecular Chemistry, C.N.R., Via Campi flegrei 34, Comprensorio Olivetti, Pozzuoli (NA), Italy. Tel.: +39081-8675018; Fax: +39-081-8041770; E-mail: [email protected] and Dr. Fabiana Piscitelli, Endocannabinoid Research Group, Institute of Biomolecular Chemistry, C.N.R., Via Campi flegrei 34, Comprensorio Olivetti, Pozzuoli (NA), Italy. Tel.: +39-081-8675309; Fax: +39-081-8041770; E-mail: [email protected]

Abstract Cannabis has been known as a medicine for several thousand years across many cultures and its beneficial effects are due to the presence of cannabinoids, unique natural products, whose pharmacology is going to gain increasing interest in scientific community. The discovery of the main psychoactive constituent of Cannabis sativa L., ∆9-tetrahydrocannabinol (∆9-THC), has led to the identification of at least one hundred additional phytocannabinoids, including cannabidiol (CBD), cannabidivarin (CBDV), ∆9-tetrahydrocannabivarin (∆9-THCV) and cannabigerol (CBG). These molecules are gaining growing interest for their medical properties, however, further research is needed to assess the differences in their pharmacokinetic and pharmacodymanic profiles. Hence, the aim of this study was to set up a rapid and accurate method, by using the LC-MS-IT-TOF technology, to detect and quantify CBD, CBDV, ∆9-THCV and CBG in biological matrices. Data 1 ACS Paragon Plus Environment

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show that the method developed here is linear in the calibration range; recoveries from mouse tissues were in the 50-60% range and sensitivity was 2 ng/ml for CBDV, 4 ng/ml for CBG and THCV and 7 ng/ml for CBD. The method is rapid, precise and accurate and it will represent a fundamental tool to evaluate the pharmacokinetic and pharmacodynamics properties of selected phytocannabinoids in tissues from different animal models and develop new cannabinoid-based medicine.

Introduction Since the discovery in 1964

3

of the main psychoactive component of Cannabis sativa L. ∆9-

tetrahydrocannabinol (∆9-THC), further research showed the presence of at least another additional 100 “phytocannabinoids” 4 . In addition to ∆9-THC, other major constituents of the Cannabis plant include: cannabidiol (CBD), cannabidivarin (CBDV), ∆9-tetrahydrocannabivarin (∆9-THCV) and cannabigerol (CBG) (Figure 1). Medical research has shown an increasing interest in the use of cannabinoids in clinical settings, particularly in patients where more conventional treatments have failed. In the last 3 decades, our knowledge of cannabinoid pharmacology has made great strides, allowing the development of novel cannabinoid-based medicines for the treatment of different human pathologies. These medicines include Cesamet® (nabilone, synthetic analog of ∆9-THC) and Marinol® (dronabinol, synthetic ∆9-THC for oral administration), which are currently prescribed in several countries to counteract nausea and emesis in cancer patients undergoing chemotherapy, and were

approved in the 90s for anorexia/cachexia associated with AIDS or chemotherapy

5

.

Futhermore, GW Pharmaceuticals have developed an oromucosal spray marketed as Sativex® (Nabiximols, a THC-CBD enriched botanical extract), which has been approved for the treatment of specific symptoms, moderate to severe (i.e. spasticity and pain) of multiple sclerosis patients in several countries 8 . The introduction of Sativex® was important for several aspects. First of all, it provided the first example of a complex botanical drug including two main active ingredients and 2 ACS Paragon Plus Environment

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approved as a single drug For instance, CBD has been introduced since it is believed to reduce the unwanted central effects of ∆9-THC. In particular, CBD may inhibit the metabolism of THC in the more psychoactive 11-OH-∆9-THC, or indirectly antagonise its actions in the brain

2, 6, 7, 11

.

Moreover, the composition of Sativex® facilitates the activation of different mechanisms and targets. In fact, ∆9-THC and, particularly, CBD may act through cannabinoid receptor-independent pathways [both activate/desensitize thermosensitive transient receptor potential (TRP) channels of vanilloid type-1 or -2 (TRPV1 or TRPV2)

17

, whereas THC mainly activates CB1 and CB2

receptors 1 . Importantly, the efficacy of this drug may be enhanced by ‘entourage’ effects of other phytocannabinoids present in the ∆9-THC and CBD extracts of which Sativex is composed

11

.

However, although the introduction of Sativex marks an important step for the medical use of cannabinoids, the use of non-∆9-THC phytocannabinoids as alternative medicines to avoid psychoactive effects remains a crucial point for pharmaceutical industries. More recently, GW Pharmaceuticals developed another lipid-based oral solution, named Epidiolex®, an investigational drug, which is composed of purified CBD currently in Phase III trials for treatment of seizures associated with Dravet Syndrome, LGS syndrome, and Tuberous Sclerosis. It has received Orphan Drug

Designation

from

the

FDA.

(http://www.gwpharm.com/GW%20Pharmaceuticals%20Announces%20Positive%20Phase%203% 20Pivotal%20Study%20Results%20for%20Epidiolex%20cannabidiol.aspx).

Therefore,

further

studies on the toxicology, pharmacology and molecular mechanisms of other non-psychoactive phytocannabinoids are required to advance cannabinoid drug development. In particular, cannabidivarin (CBDV) and ∆9-tetrahydrocannabivarin (∆9-THCV) are undergoing clinical trials for epilepsy and type 2 diabetes, respectively, whereas cannabigerol (CBG) has demonstrated positive results in animal models of inflammatory bowel disorders colon cancer

13, 19

14, 20

and against prostate and

. However, in order to perform comprehensive pre-clinical research on the

therapeutic effects of these molecules, knowledge of their pharmacokinetic properties is also necessary. 3 ACS Paragon Plus Environment

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For this purpose, we aimed at developing a rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the quantification of CBD, CBDV, CBG and THCV in mouse peripheral tissue extracts through isotope dilution mass spectrometric measurements. Furthermore, we applied this methodology to quantify colonic CBDV levels in an experimental model of murine colitis induced by 2,4,6,dinitrobenzene sulfonic acid (DNBS), immediately after treatment with CBDV 21 .

Figure 1: Chemical structures of phytocannabinoids used in this study. Materials and Methods Materials 4 ACS Paragon Plus Environment

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Reagents. CBD, CBDV, CBG and THCV, extracted from Cannabis plants, were subsequently purified [purity by high-performance liquid chromatography (HPLC): 99%] and provided by GPharm (Kent,UK). Deuterated phytocannabinoids, i.e. [2H]4 CBD, [2H]4 CBDV, [2H]4 CBG [2H]4 THCV were synthesised and provided from GW Pharmaceuticals (Porton Down, Wiltshire, UK). Methanol and water were purchased from Sigma-Aldrich and were RS-Plus grade. Acetone was purchased from VWR and was HPLC grade.

Methods Mice Male ICR mice weighing 25–30 g, were obtained from Charles River Laboratories (Calco, Lecco, Italy) and housed in polycarbonate cages under a 12-h light/dark cycle with light on at 07:00 a.m., controlled temperature (23 ± 2°C) and constant humidity (60%). Mice for experimental colitis studies were fed ad libitum with standard food, except for the 24 h period immediately preceding the administration of 2,4,6,dinitrobenzene sulfonic acid (DNBS) and for the 2 h period preceding the oral gavage of drugs. All experiments complied with the Italian D.L. no. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC).

Induction of colitis and pharmacological treatment Colitis was induced by the intracolonic administration of DNBS as per the previously described method 18 . Briefly, mice were anesthetized with inhaled 5% isoflurane (Centro Agrovete Campania, Scafati, SA, Italy) and DNBS (150 mg/kg) was injected in the distal colon using a polyethylene catheter (1 mm in diameter) via the rectum (4.5 cm from the anus). All animals were sacrificed three days after DNBS administration by asphyxiation with CO2, the mice abdomen was opened by 5 ACS Paragon Plus Environment

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a midline incision and the colon removed and stored at -80°C until use. The dose of DNBS (150 mg/kg) and the time point of damage evaluation (i.e., 3 days after DNBS administration) were selected due to data from previous studies 18 . CBDV or vehicle was given intraperitoneally (0.3, 1, 3 and 10 mg/kg) or by oral gavage (1, 3, 10 and 30 mg/kg). The last dose of CBDV was administered

1 hour (for intraperitoneal

administration) or 2 hours (for oral gavage) before the sacrifice. CBDV (IP or orally) was given once a day starting from three days before DNBS and for the following days until the sacrifice (seven days treatment in total, preventive protocol) or was given once a day starting from one day after DNBS and for the following days until the sacrifice (three days treatment in total, curative protocol). After sacrifice the colons were immediately frozen in liquid nitrogen and stored at -80°C until extraction for CBDV quantification with the same protocol reported in the “Extraction and purification from tissues” section.

Extraction and purification from mouse peripheral tissues Mice (N=25) were used for these measurements. We compared the performance of three different extraction method: ice-cold acetonitrile9, 12 , a modified Folch extraction 22 and acetone. Acetonitrile and modified Folch extraction provided a recovery less than 20% (data not shown). Pancreas and colon samples, weighing 200 mg, were dounce-homogenized in 600 µl of acetone and sonicated in an ultrasonic bath for 8 minutes. Internal deuterated standards for CBD, CBDV, CBG and THCV quantification by isotope dilution ([2H]4 CBD, [2H]4 CBDV, [2H]4 CBG and [2H]4 THCV) were added to the homogenate, which was then centrifuged (10000 rpm 1’, 4°C) and extracted at 4°C four times with 600 µl of acetone. The lipid-containing organic phase was dried down, weighed and pre-purified by open bed chromatography on silica gel. Fractions were obtained by eluting the 6 ACS Paragon Plus Environment

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column with 99:1, 90:10 and 50:50 (v/v) chloroform/methanol. The 99:1 fraction was used for CBD, CBDV, CBG and THCV quantification by LC-MS-MS analysis. Mice (N=5-6) were used to assess colonic CBDV levels in experimental model of colitis in mice. Colon samples (weighing ̴ 150 mg) were extracted as described above. The lipid-containing organic phase was dried down, weighed and pre-purified by open bed chromatography on silica gel. Fractions were obtained by eluting the column with 99:1, 90:10 and 50:50 (v/v) chloroform/methanol. The 99:1 fraction was used for CBDV quantification by LC-MS-MS analysis. LC-MS-IT-TOF analysis CBD, CBDV, CBG and THCV levels were measured by LC-MS-MS using an LC20AB coupled to a hybrid detector IT-TOF (Shimadzu Corporation, Kyoto, Japan) equipped with an ESI interface. We acquired full-scan MSn spectra of selected precursor ions by multiple reaction monitoring (MRM), extracted the chromatograms of the high-resolution [M-H]- values and used the latter chromatograms for calibration and quantification. HPLC parameters: Phytocannabinoids were separated using a KinetexC18 column (10 cm x 2.1 mm, I.D. 5µm, 100A; Phenomenex) and eluted with an isocratic flow of methanol:water (75:25) with 0.1% NH4C₂H₃O₂. LC20AB HPLC pumps were used to deliver solvent at a flow rate of 150 µl/min. The samples were injected with a SIL-20 AC autosampler (Shimadzu Corporation, Kyoto, Japan). Column oven was set at 30°C. Mass parameters: Electrosprayed ions were generated using a capillary voltage of 4.66 kV. A curved desolvation line (CDL) was set at a temperature of 250°C to aid desolvation and a heat block temperature of 220°C was also used. To help nebulisation of the electrospray solution, nitrogen was pumped into the ion source at a rate of 1.5 L/min. The ToF mass analyser was used to acquire data in both MS and MS/MS modes. In the MS mode, a 10 msec ion accumulation time was used before ion trapping. In the MS-MS mode, instead, the ion accumulation time was 20 msec and the window used for precursor ion isolation corresponds to a width of 3 amu and 20 msec. To induce 7 ACS Paragon Plus Environment

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fragmentation of the precursor ion a supplementary AC (alternative current) potential was applied to the end-cap electrodes to induce resonant excitation and argon is used as a collision gas during collision-induced dissociation (CID). The collision was carried out over 30 msec using a q value of 0.251 (45 kHz). Three scans were accumulated in each MS-MS spectrum. In both MS and MS-MS mode data were acquired over a mass range of 200-500 m/z. In both regimes of operation ions are pulsed into the ToF with an accelerating potential of 9 kV and the detector voltage is set at 1.7 kV 22 . LODs and LOQs The limit of detection (LOD) was defined as the lowest concentration with a signal-to-noise (S/N) ratio > 3. The lowest limit of quantification (LLOQ) was defined as the lowest concentration at which the analyte signal was at least ten times the signal of the blank response. To this aim, for each of the four analytes 6 standard stock solutions, ranging from 0.01 to 100 µM, were spiked in the matrix (a ~20 mg aliquot of colon tissue). LODs and LOQs were evaluated by calculating the standard deviation (SD) of each stock solution and plotting SD against the concentration to obtain S0 (Y intercepts). LOD was calculated as 3S0 and LOQ 10S0. These experiments were repeated in triplicate at three different days to evaluate inter-day and intra-day precision and accuracy (see below). Linearity and carryover Calibration standard samples were prepared in the same manner described above for LOD evaluation. For each analyte six solutions were prepared and spiked in the biological matrix (colon samples) and an aliquot of the tissue extract (5 µl) was injected in the mass spectrometer in triplicate. The amount of the deuterated standards injected into the tissue was 25 pmol, whereas the amount of the non-deuterated standards ranged from 0.05 to 1000 pmol. Calibration curves were plotted as the peak area ratio of the analyte over its respective deuterated standard against the

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concentration of the calibrator. To assess linearity, the line of best fit was determined by least square linear regression . The lack of significant carryover phenomena was verified by injecting the calibrator at increasing and decreasing concentration in triplicate, with a blank sample between each calibrator. Recovery Four pancreas samples from male ICR untreated mice were spiked with the appropriate amount of deuterated standard (2.5 µM) before extraction. Recoveries (R%) were calculated for each analyte at three concentrations of non-deuterated standard (0.02-0.2-2µM, i.e. “low”, “medium” and “high”), and were obtained by comparing the amounts of the non-deuterated standards calculated by isotope dilution before extraction (i.e. by analysing the mixtures of deuterated and non-deuterated standard solution directly injected into the mass spectrometer, A), with those calculated following analysis of the extracts spiked with deuterated and non-deuterated standard solutions (B). Accordingly, R (%)= A/B× 100. Four additional samples that did not contain any standard were processed according to the same procedure to rule out contamination problems. Precision and accuracy The intra- and inter-day precision, and the accuracy of the method for the four analytes were calculated at three concentrations (0.02-0.2-2µM) for each analyte spiked in pancreas samples, as described above. The experimental intra-day precision was expressed as CV % and the inter-day precision as relative standard error. Accuracy (Accuracy %) was calculated as the ratio between the average of the concentration measured experimentally and the nominal concentration× 100. These results indicate that the method is precise and accurate.

Results and Discussion 9 ACS Paragon Plus Environment

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Optimisation of chromatographic and mass spectrometric parameters CBDV, CBD, CBG and THCV exhibited retention times of 4.6, 8.3, 8.5 and 8.8 min respectively (Figure 2). The quantification was performed by isotope dilution by using m/z values of 289.2111 and 285.1859 and 285.1843 corresponding to the molecular ion [M-H]- for deuterated and undeuterated CBDV and THCV, respectively; or m/z values of 317.2424 and 313.2178 corresponding to the molecular ion [M-H]- for deuterated and undeuterated CBD, respectively; and m/z values of 319.2581 and 315.2296 corresponding to the molecular ion [M-H]- for deuterated and undeuterated CBG, respectively (Figure 2). Table 1 lists the retention time for each compound, the transition ions monitored, the relative molecular formula and the number of double bond equivalent (DBE). Moreover, LC-MS-IT-TOF delivers high mass resolution (10’000 at 1’000 m/z) providing mass values with four decimal places. To the best of our knowledge, this method is the first described method allowing the simultaneous determination of these 4 compounds in one sample preparation and injection.

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Figure 2: Targeted profiling of phytocannabinoids in lipid extracts using LC-MS-IT-TOF. Representative extracted ion chromatogram of a pre-purified pancreas lipid extract containing CBDV, CBD, THCV and CBG with the relative high-resolution MS spectra of the four compounds.

Table 1. MRM transitions and their corresponding molecular formula and DBE.

Validation 11 ACS Paragon Plus Environment

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Linearity, LODs and LLOQs The ratio between the [M-H]- peak areas of non-deuterated (0.05-1000 pmol) vs. deuterated (25 pmol) compounds varied linearly with the amount of non-deuterated compound (Figure 3). The calibration curves were found to be linear in the calibration range and the equations and correlation coefficients (R2) were determined. LODs and LLOQs for CBDV, CBD, THCV and CBG are listed in Table 2.

Figure 3: Calibration curves for CBDV, CBD, THCV and CBG. The correlation coefficient (R2) and equation is shown for each curve.

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Table 2. LOD and LOQ values for Phytocannabinoids. Compound CBDV CBD THCV CBG

LOD (ng/ml) 2.2 7.1 4.3 4.7

LOQ (ng/ml) 7.5 23.7 14.2 15.8

Recovery and precision Recoveries are listed in Table 3 for the four analytes, and were comparable to those of previously reported methods 10 . Intra-day and inter-day precision data are also summarized in Table 3 and data are expressed as CV (%) and relative standard error, respectively.

Table 3. Recoveries, intra-day, inter-day precision and accuracy data. Compound

CBDV

% Recovery (%CV)

% Matrix effect (%CV)

Accuracy (%)

0.02

58.9 (4.7) 64.9 (1.8)

41.6 (5.7) 56.9 (4.4)

99 98

3.0 1.2

0.02 0.01

60.1 (3.8)

47.5 (6)

99

4.6

0.03

86.8 (1.2) 81.6 (4.7)

74.3 (7) 40.3 (3.4)

99 99

8.2 15.2

0.05 0.09

0.2

2 0.02 CBD

THCV

CBG

Precision Inter-day (relative standard error)

Concentration (µM)

0.2

Intra-day (%CV)

2 0.02

68.9 (2.5)

35.6 (1)

99

1.9

0.01

86.2 (4)

78.9 (5.7)

7.7

0.04

0.2

81.0 (6)

71.0 (6)

5.9

0.03

2 0.02

98.6 (9)

74.7 (9.3)

7.8

0.05

59.9 (2.1)

42.5 (5)

12.9

0.08

0,2

67.6 (3.2)

58.2 (3.8)

1.9

0.01

2

42.5 (6.9)

30.6 (2)

99 98 99 99 98 98

4.2

0.02

CBDV levels in the colon of mice with colitis No isomerization of CBDV in THCV was observed in this tissue.

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As shown in Fig.4, CBDV levels in the colon of DNBS-treated mice, following curative ip treatment with the compound, were dose-dependently higher with increasing doses of CBDV and the most significant increases in levels were observed with the maximal tested dose of 10 mg/kg dose of 10 mg/kg (P