In Vitro Release Testing (IVRT) Study of Active Ingredients, cannabidiol (CBD), from Semisolid measured with Franz Cell Diffusion Apparatus and with meta-analysis for therapeutic use. [Credits, Dr. Tarek Bader, Marek Jasinski]
July 5th, 2022
Abstract: Cannabidiol (CBD) is non-psychoactive cannabinoid isolated from Cannabis sativa, that has recently received significant attention due to its therapeutic potential and benefits . The main purpose of this study was to investigate the skin permeability of CBD semisolid emulsion provided by Sweet Earth through the skin by determining time and quantity required to reach to maximum absorption of CBD using Franz Cell diffusion apparatus. This could provide valuable information to optimize the development of topical CBD semisolid emulsion for a therapeutic use. In Vitro Release Testing studies were conducted using Franz diffusion apparatus with cellulose Nitrate membranes to determine the skin permeability for CBD semisolid emulsion at selected time points. High-Performance Liquid Chromatography (HPLC) was used to quantify CBD at 0.5, 1, 2, 3 and 4 hours. Our results show that CBD permeability begins within 30 minutes, and steadily increases to reach the maximum absorption at 3 hours. Therefore, the topical administration of CBD semisolid emulsion, from Sweet Earth, can be an optimal choice for delivering significant quantity of CBD for therapeutic purposes. Meta-analysis of selected publications indicated therapeutics potential in arthritis, muscle recovery, muscle training and performance on exercise.
Cannabidiol (CBD) is non-psychoactive cannabinoid isolated from Cannabis sativa. Although, Cannabis sativa has a long history associated with medicinal use [12,13], recent studies have shown that CBD’s usage has been significantly increasing in the last several years due to its therapeutic and psychoactive properties. Furthermore, the medical usefulness of cannabinoids has been more rigorously studied and its anti-inflammatory effects were demonstrated [2,4,6,14,16,37]. Consequently, CBD skin products, including creams, gels, and oils have been extensively used in the last decade for its therapeutic effects. Scientists continue to explore the 2 effectiveness of CBD in a range of medical conditions, including Parkinson's disease, schizophrenia, diabetes, multiple sclerosis, anxiety and different types of cancer [2,4,6,7,11,18,24,27,37]. Topical/transdermal CBD delivery has advantages over other routes since it is the most preferred route for dermatologists and patients due to its localized effect of a specific area while not affecting other organs . Despite the extra focus on CBD as a topical treatment, there is very limited research on the permeability of CBD through skin, resulting in lack of the experiential evidence on CBD permeability of cosmetic products. As pre viouslyindicated, the main purpose of this study is to investigate the accumulative absorption of CBD from Sweet Earth CBD cream through the skin by determining time and quantity required to reach to maximum absorption of CBD using Franz Cell diffusion apparatus. This could provide beneficial evidence for the manufacturer to develop a suitable CBD delivery system that could be used in advancing their topical products.
Materials and Methods
CBD semisolid emulsion, which contains 6.08725mg of CBD per ml, was provided by Sweet Earth LLC, Ashland, United States. Franz diffusion apparatus, Cat # 2351, was purchased from Alfa Engineering Solutions, KSHITU R&D LAB Instruments, cat no: 2351 (Thane, Maharashtra, India). Cellulose Nitrate membranes (Global Life Sciences Solutions Operations UK Ltd.) catalogues number 7184-002, lot number: A29574748, with a pore size of 0.45 µm, diameter 25 mm was purchased from GE Healthcare Life (Global Life Solutions Operations UK Ltd, Buckinghamshire UK). Hexane HPCL Plus, Cat650551, was purchased from Sigma Aldrich system, Merck SA, An affiliate of Merck KGaA, Darmstadt, Germany.
a. Study Optimization:
The experimental conditions in this study are based on the literatures, a consultation with an expert in the field of skin permeability and pretesting experiment. Moreover, a great amount of work has been completed in our laboratory to optimize this study, we have tested different conditions, such as type of solvent vs no solvent, solubility of the cream, membrane compatibility, time points and 3 the length of the study to ensure that the ideal experimental conditions were met before proceeding to the actual experiment. Sweet Earth cream is an oil-based product and various solvents have been tested to determine the solvability of the cream. These solvents include hexane, methanol and butanol in addition to no solvent in the donor compartment. Two types of membranes were tested, Start-M and Cellulose Nitrate membranes. Cellulose Nitrate membranes showed a higher permeability for the tested cream and has a high compatibility with hexane unlike other solvents. Therefore, hexane was selected as a collection buffer in the receiver compartment. In terms of ength of time and point of the sample’s collections, we have tested a wide range of time points, 30 minutes, 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours. The pretesting experiment showed that the permeability starts within 30 minutes post application and reaches its peak at around 3 hours. No significant increase in cream absorption occurs from 5 to 24 hours, despite the consistent cream availability, which is mainly due to the membrane saturation. Based on these results we proceeded to design a real world like study to measure the absorption of effective CBD that may be available for use once absorption through the skin occurs. Therefore, time points 0.5, 1, 2, 3 and 4 hours were selected to perform the experiment in duplicate.
b. In Vitro Skin Permeation Study
Franz diffusion cells apparatus is available in our laboratory for this testing. We used Sweet Earth CBD cream while synthetic membranes (Nitrocellulose, Cat # 7184-002, lot # A29574748, 0.45um, diameter, 25mm GE Healthcare Life) were used as model barriers. The investigated parameters included time and quantity of CBD. The in vitro permeation studies were performed under non-occluded conditions by using Franz diffusion cells with a permeation area: 1.77 cm2; receptor chamber volume: 10 mL, and the use of Cellulose Nitrate membranes. In this experiment, samples of 1.4 grams of Sweet Earth cream, contains 8.52 mg CBD/each, were loaded directly in the donor compartment of each cell. Cellulose Nitrate membranes were oaked, according to manufacture instructions in 100 % hexane for 30 minutes prior to assembling the diffusion cells. Receptor compartments were filled with 10 ml of 100 % of Hexane, in which the solubility of CBD was predetermined, and the hexane showed high compatibility with Cellulose Nitrate membranes’ permeability. A micro spatula was used to apply CBD semisolid emulsion as finite (non-occluded condition) to the membranes and the applied 4 formulation was spread evenly using the back side of a spatula. Special care was taken to avoid the formation of air bubbles between the membrane and the hexane in the receptor compartment. Furthermore, before the two parts were fastened together using a clamp, the gap between upper and lower compartments of the vertical-type Franz cells was sealed with a thin layer of Vaseline at the outer edge of the cell. Also, the sampling port and the opening of the donor compartment were covered with a plastic wrap to prevent the evaporation of hexane. The system was kept at 37 ◦C by a circulating water bath, which resulted in the membrane surface temperature being 32 ± 1 ◦C. The stirring speed was 500 rpm throughout the experiment. All these conditions were maintained throughout the experiment. At predetermined times 0.5, 1, 2, 3 and 4 h, the entire volume of 10 ml of hexane was withdrawn from the receiver, and stored at 4 ͦC until a High-Performance Liquid Chromatography (HPLC) analysis was completed by Sigma Analytical Services Inc (Toronto, Canada). [1,3,5,7,9] C. High-Performance Liquid Chromatography (HPLC) HPLC was completed by Sigma Analytical Services Inc (Toronto, Canada). “HPLC analysis was carried out by using the PerkinElmer Flexar™ HPLC system, manufactured by Terra Universal, Inc 800 S. Raymond Ave., Fullerton, CA 92831, and equipped with photodiode array (PDA Plus™) detector. The experimental conditions were as follows: C18 column, mobile phase - gradient of Mobile Phase A and B (27/ 73 V/V%). A: ammonium formate/water (441.39 mg in 1L) and B: 1.00mL of formic acid in acetonitrile to total volume of 1000mL.
Table -1- summarizes the results of the duplicate experiments for the cumulative quantity of CBD (mg) released from Sweet Earth CBD cream through Cellulose Nitrate membranes. Our study shows a steady increase in CBD permeability that begins before 30 minutes and reaches the maximum absorption at 3 hours, then followed by a slight reduction in the absorption in CBD at 4 hours. Moreover, the total accumulative CBD (mg) at different testing points of 0.5, 1, 2, 3 and 4 hours was calculated and plotted with time. Figure-1- shows the total accumulative amount of 5 CBD (mg) at 0.5, 1, 2, 3 and 4 hours, which provides the total quantity of CBD passing through cellulose nitrate membrane at each given time point using 1.4 g cream for each time point.
Table-1-The cumulative amount of CBD (mg) released from Sweet Earth CBD cream through Cellulose Nitrate membranes at different time points.
Figure-1- shows the total accumulative amount of CBD (mg) at 0.5, 1, 2, 3 and 4 hours.
These results shows that CBD absorption started before 30 minutes and followed by a steady increase at 1, 2, and 3 hours, then a slight decline at 4 hours. Our calculations are based on HPLC analysis for the 10 ml of hexane in the receiver compartment, which were collected at each time point. Consequently, we have calculated the total of CBC passed through the membrane from 1.4 g cream (contains 8.52 mg CBD). Our results demonstrate that at 0.5 hour, the accumulative CBD quantity is 6.7 % of the total CBD loaded on the donor compartment, at 1 hour is 10.2 %, 2 hours 10.9 %, 3 hours is 15.3 % and 4 hours 13 %. Our results suggests that Sweet Earth CBD cream is a practical method to deliver CBD into a tissue through the topical application. Furthermore, our results demonstrate that the transdermal administration provides a steady CBD infusion to be delivered over a continued period of time, while also reducing the side effectsof higher drug peak concentrations.
The topical administration of Sweet Earth’s CBD cream shows optimal cumulative absorption time at 3 to 4 hours. Consequently, the main active ingredient (CBD) and the small amounts of Cannabigerol (CBG) are effective for improvement of quality of life in people with different chronic inflammatory diseases. Our meta-analysis based on time penetration evaluation has revealed its positive effects on osteoarthritis and general arthritis related effects [16,17,24,27,55]. Additionally, the analysis revealed a potential role in muscle building. Muscle tissue recovery time and performance is evaluated based on timed HPLC analysis. CBD has a high affinity to G-protein-coupled receptors Figure (2) GPR3, GPR6, GPR12, GPR18, GPR55, GPR119  and peroxisome proliferator-activated receptors (PPAR-α and PPAR-γ) [19,21, 22]. This affinity increases anti-inflammatory eicosanoids production, making the compound a target for therapeutic studies with modulating compounds like DMSO to obtain consistent results .
Figure 2. CBD selective GPR map
We postulate that cumulative CBD concentration buildup is favorable for general Arthritis  and specifically Osteoarthritis (OA) [16,17]. Existing therapies for osteoarthritis (OA) management are symptom reliving strategies only. These are inadequate for long term use due to associated side effects , making CBD a potentially superior avenue. This suggests CBD at 30 min mediates TRPA1, TRPV1 and TRPV4 ionotropic receptors as reported [25,26,28]. The synergistic effects of DMSO inhibition of Histamine [17,23] is well explained, however the Methylsulfonylmethane (MSM) in the cream likely attenuates the actions in TRPA1, TRPV1 and TRPV4 alongside with CBD show the impact on nitric oxide (NO) reduction and its protective effects on macrophages. This explains protective actions of CBD and is a route for further exploration in joint inflammation that could turn a corner for novel therapeutics. Combination of ingredients like MSM with its main constituent CBD have a suppressive effect on IL-1β and TNF-α [27,32] to protect cartilage [29,30,31]. These synergistic actions appear to attenuate performance of CBD.
Current research shows improving exercise performance by CBD application is sufficient when applied thirty minutes prior to exercise . Here we stipulate a similar effect if not better is likely to be achieved. CBD is an agonist for CB2 (the cannabinoid type 2 receptor) [34,35,36] and is involved in the signaling reward process, and Anandamide [AEA] in muscle cells [37,38]. Existing studies [39,40] suggest topical creams and rubs have an effect on muscle performance when applied prior to which attenuate AEA.
Figure 3. Major contributing factors affected muscle damage
Athletic exercise can lead to induced muscle damage [41,43] which has further implications for future performance [10,42]. The four major contributors that every athlete must consider on amdaily basis to continue improving performance are shown in figure-3. Due to the vast amount of data being made available, CBD is quickly gaining notability among athletes and is displacing other products  making it the fastest growing topic of interest. Athletic performance and muscle recovery is directly affected by type of exercise and the amount of induced muscle damage  in proportion to time. Exercise increases blood flow and oxygenation which produces proteins like carbonyl (PC), lipids [48,49,50] and DNA [46,47]. CBD has been shown to be a potent CB2 receptor antagonist suggesting inhibition effects of lipopolysaccharides (LPS) decrease inflammation thus reducing oxidative stress. The addition of MSM in the cream has synergistic effects on inflammation and protective actions [51,52,53,54].
As reports in clinical trials on CBD alone can be controversial [55,57], products like Sweet Earth's cream offers attenuating CBD actions which can deliver a consistent result based on synergistic cofactors like Coconut oil , MSM [29,30,51] and other ingredients. These cofactors have an impact on CBD based products. These combined actions provide evidence for development of novel based therapeutics.
The topical administration of Sweet Earth CBD cream has shown a steady skin permeability through the nitrocellulose nitrate membrane. These results show the optimal skin permeability of Sweet Earth CBD cream that occurs within 0.5 to 4 hours; therefore, the active ingredient (CBD) reaches its maximum effectiveness when applied topically within 3 hours. This suggests that topical administration is an ideal method to deliver CBD cream for therapeutic purposes. Therefore, CBD could be considered as an alternative for improvement in the quality of life for people with chronic disease such as arthritis, multiple sclerosis and other inflammatory musculoskeletal diseases [2,3,4,24,27,33,37]. Furthermore, it is clear CBD will continue to play a major role in athletic performance.
Acknowledgement of funding:
This work was supported by:
Sweet Earth LLC,
258 A Street #469, Ashland, OR United States 97520, USA
NAVCO Pharmaceuticals Limited
5205 Harvester Rd. Unit 1
Burlington, Ontario, L7L6B5, Canada
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