Effect of Quantitative Structural Properties and Drug Formulation in Four Cannabinoids (Cannabidiol, Cannabigerol, Cannabichromene, and Cannabinol) on Their Lymphatic Transport after Enteral Administration in Rats

Abstract

The effect of quantitative structural properties of drugs on the extent of lymphatic transport is not well understood. Our study aimed to describe these principles in four cannabinoids, cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN) administered as oil solutions and nanoemulsions. A series of studies in jugular vein cannulated rats and anesthetized mesenteric lymph duct cannulated rats was conducted to measure drug oral bioavailability and lymphatic transport. Log P was measured, and quantitative structural properties were correlated to the extent of lymphatic absorption. Nanoemulsion did not increase the absolute bioavailability via lymph in CBD but led to an 8-fold increase in CBG and a 3-fold increase in CBC and CBN. There was an even higher increase in the absolute bioavailability via portal vein (11-fold for CBD, 71-fold for CBG, 8-fold for CBC, and 13-fold for CBN). Relative bioavailability via lymph increased with decreasing smallest orthogonal molecular size and topological polar surface area. Nanoemulsion did not affect the total oral bioavailability but led to an increased absorption into portal blood. Intestinal lymphatic transport plays a major role in the absorption of CBD, CBG, CBC, and CBN. Planarity of the molecule and low surface polarity could be crucial structural features facilitating lymphatic transport.

Introduction

Lymphatic transport of drugs after oral administration plays an important role in the absorption of highly lipophilic compounds with log P > 5. (1−4) After solubilization in the gastrointestinal tract, these compounds are absorbed into enterocytes, the intestinal epithelial cells, where they are incorporated into chylomicrons. Chylomicrons are large lipoprotein particles produced specifically in the intestines that are naturally involved in the transport of highly lipophilic molecules. Their size/diameter is too large to pass into the intestinal blood capillaries. Therefore, they take an alternative route via the intestinal lymphatic system. Chylomicrons enter the lacteals (lymph capillaries) and are transported through a network of lymphatic vessels and mesenteric lymph nodes until they reach the thoracic duct, where intestinal lymph mixes with lymph from the lower parts of the body. All the lymph eventually enters the systemic blood circulation at the confluence of the thoracic duct with the jugular and subclavian veins.

The transport through the intestinal lymphatic system can have a major impact on the drug pharmacokinetics. It can increase the absolute oral bioavailability because the lymph represents an additional gateway into the systemic circulation besides the standard transport through the portal vein. Another mechanism for increasing oral bioavailability is by avoiding first-pass metabolism in the liver. Mesenteric lymph is a dominant (sometimes even exclusive) source of systemically available drug for compounds with a high extraction ratio in the liver. (5,6) Lymphatic transport can improve the efficacy of drugs that act against components of the lymphatic system, such as immunosuppressants, anti-HIV and anticancer drugs by reaching high exposure in the mesenteric lymphatic system. (7,8)

Given the possible advantages of targeting drugs into the mesenteric lymph, various approaches have been tested to increase lymphatic transport. However, summarizing all the available literature on in vivo lymphatic transport data, it seems that different strategies must be implemented based on the drug’s lipophilicity. In highly lipophilic compounds (log P > 5), it may be sufficient to use a lipid-based drug formulation (oil solution, o/w emulsion, self-nanoemulsifying system, etc.), because lipids (especially long-chain triglycerides) promote the physiological assembly of chylomicrons in which the drugs are dissolved and transported through the lymph. (9,10) Targeting drugs with lower lipophilicity (log P < 5) into the mesenteric lymph is much more challenging. The only functional technique discovered so far is a synthesis of a lipophilic prodrug by covalently binding a lipid residue to the original drug molecule. Such complex molecule has log P > 5 and is effectively transported into the lymph. Examples of drugs successfully targeted into the lymph despite their insufficient lipophilicity are e.g. mycophenolate, valproate, and paracetamol. (11−13) There are few reports on targeting low-lipophilicity drugs into the lymph without lipophilic prodrug synthesis. However, the extent of the lymphatic transport for these compounds has been investigated using a noninvasive lymphatic transport measurement, which has been shown to significantly overestimate the results. (14−16)

Cannabinoids are an important group of compounds found naturally in cannabis or produced synthetically and frequently used as registered medicines, nutritional supplements or abused as recreational drugs. They are lipophilic, mostly with log P > 5, and are therefore suitable model drugs for lymphatic transport driven absorption. The most frequently investigated cannabinoids are THC (tetrahydrocannabinol), the main psychoactive compound in cannabis, and CBD (cannabidiol), the second most abundant but nonpsychoactive cannabinoid. Besides these two, there is a variety of “minor” cannabinoids with much less pharmacological data available. In this work, we focus on four nonpsychoactive compounds: the “major” cannabinoid CBD, and three “minor” cannabinoids CBG (cannabigerol), CBC (cannabichromene), and CBN (cannabinol). Their structures are shown in Figure 1.

Figure 1. Structures of the four tested cannabinoids: cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN) — **Figure 1.** Structures of the four tested cannabinoids: cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN)

CBD has been registered for the treatment of specific forms of childhood epilepsy. It is administered orally in a formulation based on sesame oil solution (Epidyolex). Besides that, CBD, together with THC, are the active components of authorized cannabis extract registered for the treatment of spasticity in patients with multiple sclerosis (Sativex). CBD has a very limited oral bioavailability, estimated at <10% in man. (17) When taken with food, the bioavailability increases up to 4-fold. (18) Preclinical pharmacokinetic data are consistent with the human observations: CBD had a bioavailability of 8% and 22% in rats when administered in a lipid-free formulation and in a long-chain triglycerides-based formulation, respectively. (19)

CBG is a much less known cannabinoid compared to CBD. There are no registered drug products containing this compound worldwide. Nevertheless, it is marketed as a nutritional supplement and various pharmacological effects have been reported in preclinical studies. CBG possesses antioxidant, anti-inflammatory, and antitumoral activities, and has antianxiety, neuroprotective and appetite-stimulating effects. (20) Absolute oral bioavailability is not known. When administered to dogs, CBG was rapidly absorbed with an average Tmax of 0.75 h. (21) When coadministered with food, the speed and extent of absorption did not change significantly.

CBC has shown an anti-inflammatory and antihypertensive effect in preclinical studies. (22,23) After oral administration to man, it absorbs with moderate speed (Tmax 2–4 h). Absolute oral bioavailability is not known. (24)
CBN was successfully tested in the treatment of dermatological disease epidermolysis bulosa. (25) After oral administration to rats, it was absorbed with Tmax of 1.5 to 3 h. (26) Absolute oral bioavailability was not determined.
One way of increasing the bioavailability of lipophilic compounds with problematic dissolution in the gastrointestinal tract is the development of formulations where the active substance is dispersed in small lipid droplets (microemulsions, nanoemulsions, self-emulsifying systems etc.). This method was successfully used e.g. in ivacaftor, where the bioavailability was increased 7-fold in Beagle dogs and previously present positive food effect was eliminated. (27) There are discussions, whether this bioavailability increase is solely due to better solubilization of the active substance in the gastrointestinal tract, or if there are some additional mechanisms like e.g. involvement of the intestinal lymphatic transport. Unfortunately, there are only very few studies comparing the lymphatic transport of one particular compound administered in a basic formulation (aqueous or oil solution) and in an advanced formulation containing a solubilized drug. (2) In our recently published preliminary data, the CBD oral bioavailability did increase mainly due to increased nonlymphatic transport when administered as a microemulsion compared to a simple oil solution. (28)

There are specific physicochemical properties and molecular structure characteristics that help the drugs partition into the lymph after intestinal absorption. The role of log P has been postulated already several decades ago by Charman and Stella. (1) The same authors also discussed the importance of compound solubility in long-chain triglycerides. However, at least one molecule was later found (penclomedine) with a very limited partitioning into lymph despite high lipophilicity (log P > 5) and high solubility in long-chain triglycerides (>50 mg/g). (29) Moreover, log P was absent in the mathematical model developed by Holm and Hoest predicting precise lymphatic transport of drugs that was based on in vivo data from 19 molecules. (30) Instead, factors such as the size of the hydrophilic area in the molecule and hydrophilic–lipophilic ratio did play a role in this model. It is therefore evident that no single physicochemical property or structural pattern can be used to precisely predict the extent of lymphatic transport and the result is formed by an interplay of more variables. Unfortunately, there is a lack of experimental data investigating molecular structure characteristics like molecular size, molecular shape, and surface polarity on the extent of lymphatic transport.
The aim of this study was to evaluate whether an advanced drug formulation (a nanoemulsion) increases bioavailability via portal blood or via lymphatic transport and to assess the importance of quantitative structural properties of the molecule for the extent of lymphatic transport. To achieve these aims, we investigated four similar compounds CBD, CBG, CBC and CBN and their quantitative structure–activity relationship (QSAR) with lymphatic transport after oral dosing in a simple formulation (oil solution) and a nanoemulsion.

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Materials

In the pharmacokinetic and lymphatic transport studies, the cannabinoids CBD, CBG, CBC, CBN (purchased from Pharmabinoid, Netherlands) were administered in sunflower oil and nanoemulsion. The required amount of cannabinoid was dissolved in sunflower oil of pharmaceutical quality (Fagron, Czech Republic) to obtain a reference formulation. The composition of the nanoemulsion formulation was optimized. In brief, a three-component system was selected─propylene glycol monocaprylate (PGMC; Gattefosse’, France), Kolliphor EL (CR-EL; BASF, Germany) and the third component acting as a cosurfactant and solubilizer was subject to optimization. Diethylene glycol monoethyl ether (TRSC; Gattefosse’, France), ethyl alcohol – 99.8% (ETOH; Penta, Czech Republic) or propylene glycol – 99.5% (PRGL; Penta, Czech Republic) were tested as cosurfactant and solubilizer. According to the optimization results and literature research, TRSC was chosen for further in vivo experiments. Further information regarding optimization is shown in the Supporting Information. The nanoemulsion formulation used in the pharmacokinetic study for both the oral and the intravenous administration was prepared as follows. Cannabinoid was dissolved in the oil phase (50 wt % of CR-EL, 30 wt % of TRSC and 20 wt % of PGMC). Next, four parts of water with respect to one part of oil (by weight) were added dropwise to the oil mixture at mild stirring. The final formulation was kept out of light and stored in the fridge before application.

Pavel Ryšánek, Petr Jelínek, Hynek Housar, Petr Kozlík, Tomáš Křížek, Anežka Nováková, Michaela Sklenárová, Viktória Paulusová, Sara Merdita, Mahak Arora, Olesia Symkanych, Monika Šteigerová, Eliška Zmeškalová, Ondřej Slanař, Miroslav Šoóš, and Martin Šíma, Effect of Quantitative Structural Properties and Drug Formulation in Four Cannabinoids (Cannabidiol, Cannabigerol, Cannabichromene, and Cannabinol) on Their Lymphatic Transport after Enteral Administration in Rats, Molecular Pharmaceutics Article ASAP, DOI: 10.1021/acs.molpharmaceut.4c01357