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Assessing Cannabinoid Permeability in a Mouse Dermal Administration Model
D.B. Colagiovanni, PhD, DABT®, K. Chupka, MS, S. Schwarz, AB and P.H. Johnson, PhD
Next Frontier Biosciences
, Denver, CO USA
Abstract
Plasma and Tissue THC and Metabolite Data
Overall Conclusions
Relevant Literature
Study Methods
Experimental Design
Contact info: Dr. Dorothy Colagiovanni
dot@nextfrontierbio.com
www.nextfrontierbio.com
Property of Next Frontier Biosciences
Topical formulations were prepared without permeation enhancers
(NFB-A) or with permeation enhancers (NFB-B and NFB-C). On study day -1, CD-1 male mice (12-15/group) designated for topical treatments with NFB-A, NFB-B and NFB-C had the hair on their entire backs
shaved and tape stripped. Animals with skin damage were moved to the IV dose group. On study day 0, animals were randomized into
treatment groups based on body weight. Following randomization, either IV treatment with NFB-A or dermal treatment with NFB-A, -B or -C was initiated as indicated in TABLE 1. IV doses were administered based
upon weight while dermal applications were the same for all mice. Blood collection time points are outlined in TABLE 2. At final time points,
animals were anesthetized with Isoflurane and bled for blood collection and dermal punch biopsies were collected. Blood was processed to
plasma in trifluoracetic acid to prevent glucuronidation. Tissues were homogenized and brought up in 200 uL PBS for analysis.
Animal Study Design
•
SPE extraction methods enabled isolation of THC and its
metabolites
•
Plasma and tissue THC,THC-OH and THC-COOH were
analyzed by TOF LC-MS analysis with LOQ of 1, 5 and 5 ng/mL,
respectively.
•
Dermal administration of our various topical preparations
resulted in systemic exposure (<100 ng/mL) with substantial
body surface area application.
•
Permeation enhancers appear to improve dermal absorption of
THC in mouse skin samples.
Plasma Results from Groups 2-4 Dermally Applied Formulations
Cannabinoids are extremely lipophilic substances with LogP values >5. This makes cannabinoids amenable to formulation only with hydrophobic preparations for topical administration. We sought to evaluate various oil based preparations of Δ9-tetrahydrocannabinol (THC). The starting
formulation (base) did not have permeation enhancers while other
formulations did. Preparations were analyzed by liquid chromatography (LC)-mass spectrometry (MS) to confirm potency and chemical identity prior to study initiation.
Male CD-1 mice were used in the study following guidelines of humane animal use. Animals were anesthetized, dorsal regions were shaved and then tape stripped. A 200 mg sample of 0.1% THC preparation was applied to the skin of each animal. Mice were observed for signs of clinical toxicity and blood was collected by cardiac puncture 5 minutes to 4 hours
post-dose under anesthesia. After blood collection, animals were humanely euthanized. Punch biopsies of the skin were then collected from each animal. Blood samples were processed to plasma and samples were
treated with trifluoroacetic acid to prevent glucuronidation. Skin samples were homogenized and processed for tissue bioanalysis.
Plasma and tissues samples were analyzed for THC as well as the
metabolites (±)-11-Hydroxy-Δ9-THC and (-)-11-nor-9-Carboxy-Δ9-THC by TOF LC-MS. Results of the analysis demonstrated the ability to separate THC from its metabolites in biological samples and individually quantify
them from <1ng/ mL up to 500 ng/mL. The study enabled evaluation of both
in vivo skin permeation and plasma concentrations of THC and its
metabolites.
Presented at the ICR Conference of 2017, Pueblo, CO
FIGURE 6: Mean IV Plasma Data of THC and Metabolites
Solid Phase Extraction (SPE) Procedure: Plasma and tissue samples
were processed using a method developed by Waters1 and modified for our
specific study requirements. Samples were mixed with 200 uL acetonitrile to 100 uL tissue or plasma. Samples were then sonicated and centrifuged at 15K rpm for 5 min. Supernatant was then transferred to an SPE plate utilized for low volume applications containing 400µL of 0.1M NH4OAc and plate loaded. Samples were washed 2X with 100µL 75:25 H2O/MeOH. Samples were then eluted 2X with 50µL 90:10 ACN/MeOH. Plasma and tissues samples were analyzed for THC as well as the metabolites (±)-11-Hydroxy-Δ9-THC and (-)-11-nor-9-Carboxy-Δ9-THC by TOF LC-MS. The LOQ for THC was 1 ng/mL, while it was 5 ng/mL for the two metabolites.
1) Quantitative Analysis of THC and its Metabolites in Plasma using Oasis PRiME HLB for Toxicology and Forensic Labs. Zhang, Danaceau and Chambers. 2016 Waters Corporation; Application Note * We would like to thank Jed Pheneger for his technical expertise during the study
10 µL of SPE extract is injected on a 3.0 x 50 mm, 2.5 µm Waters CSH C18 column followed by TOF/MS detection
FIGURE 2: 50 ng/mL THC and Metabolite Standards in Plasma
Group An. # Time-point(s)
1 1-3 5 min 1 4-6 15min 1 7-9 1hr 1 10-12 2hr 1 13-15 3 hr 2, 3, 4 1-3 30 min 2, 3, 4 4-6 1hr 2, 3, 4 7-9 2hr 2, 3, 4 10-12 8hr
TABLE 2. Mouse Bleed Times
FIGURE 1. Representative Calibration Curves with Standards
FIGURE 3. Plasma THC Comparison FIGURE 4. Plasma THC-OH Comparison FIGURE 5. Plasma THC-COOH Comparison
FIGURE 7. Dermal THC concentrations with Topical Preparations: Gr 1-4
*note- no THC-OH or THC- COOH was detectable in the skin samples
Graphic of SPE PRiME HLB µElution plate system from Oasis used for Extraction
