Intestinal lymphatic transport of cannabinoids: implications for people with autoimmune diseases and immunocompromised individuals

There has been an escalating interest in the medicinal use of Cannabis sativa in recent years. Cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), the main constituents of Cannabis sativa, have well documented immunomodulatory effects in vitro and following administration of high doses to animals. However, these effects have not been clearly evident in humans following oral administration of cannabinoids, probably due to low systemic bioavailability. To note, cannabis and cannabis-containing medicines are currently used for symptomatic relief in autoimmune diseases, such as multiple sclerosis (MS), and in cases of immunodeficiency, such as in cancer patients on chemotherapy regimens. In this thesis, we aimed to elucidate the impact of enhancing the transport of orally administered cannabinoids to the intestinal lymphatic system, the major host of immune cells, on the immunomodulatory effects of cannabinoids. Oral administration of lipophilic cannabinoids with long-chain triglycerides (LCT) was investigated as a simple approach to enhance the intestinal lymphatic transport. The effect of LCT on the intraluminal processing of orally administered cannabinoids was assessed by means of in vitro lipolysis model. The results of in vitro lipolysis demonstrated that at least one-third of CBD dose would be solubilised and readily available for absorption to the enterocytes when orally administered in LCT-formulation. The association of CBD with chylomicrons (CM) in the enterocytes and subsequent intestinal lymphatic transport was estimated using an in silico model, in vitro association by artificial CM-like lipid particles, and ex vivo uptake by plasma-derived CM from rats and humans. The results of CM association studies revealed high intestinal lymphatic transport potential for CBD in rats and humans. Similar high lymphatic transport potential was also reported for THC in our laboratory. Oral co-administration of CBD and THC with LCT to rats increased the systemic exposure by 3-fold and 2.5-fold, respectively, compared to lipid-free formulations. The underlying mechanism of increased bioavailability is likely to enhanced intestinal lymphatic transport and decreased pre-systemic metabolism in the liver. The results of biodistribution experiments indicated that the intestinal lymphatic transport of CBD and THC was, indeed, enhanced following oral co-administration of lipids as denoted by the dramatic increase in the concentrations recovered in MLN and intestinal lymph. The concentrations of CBD and THC in intestinal lymph fluid were in the range of 120 and 60 µg/mL compared to 0.5 and 0.6 µg/mL in plasma, respectively. Moreover, CBD and THC showed dose-dependent immunosuppressive effect on lymphocytes isolated from rats and peripheral blood mononuclear cells (PBMC) isolated from humans as assessed by lymphocyte proliferation assay and flow cytometry analysis of inflammatory cytokines. These effects were only significant at concentrations achieved in the intestinal lymphatic system, but not in plasma, following oral co-administration of cannabinoids with LCT. CBD showed more immunosuppressive effects on lymphocyte proliferation and the expression of inflammatory cytokines comparing to THC. Also, PBMC from MS patients were more susceptible to the immunomodulatory effects of cannabinoids than PBMC from healthy volunteers and cancer patients on chemotherapy. In conclusion, oral administration of cannabinoids with lipids can enhance the intestinal micellar solubilisation and augment the systemic exposure to cannabinoids by enhancing intestinal lymphatic transport. The concentrations of lipophilic cannabinoids recovered in the intestinal lymphatic system were extremely high and exceeded the immunosuppressive threshold of CBD and THC. The increase in systemic exposure to cannabinoids in humans is of potentially high clinical importance as it could turn a barely effective dose of orally administered cannabis into highly effective one, or indeed a therapeutic dose into a toxic one. In addition, administering cannabinoids, in particular CBD, with a high-fat meal, as cannabis-containing food, or in lipid-based formulations could represent a valid therapeutic approach to improve the treatment of MS, or other T cell-mediated autoimmune disorders. However, in immunocompromised patients, administration of cannabinoids in this way may deepen the immunosuppressive effects

Predicting intestinal and hepatic first-pass metabolism of orally administered testosterone 3 undecanoate 4

The bioavailability of orally administered drugs could be impacted by intestinal and 12 hepatic first-pass metabolism. Testosterone undecanoate (TU), an orally administered ester prodrug 13 of testosterone, is significantly subjected to first-pass metabolism. Yet, the individual contribution 14 of intestinal and hepatic first-pass metabolism is not well determined. Therefore, the aim of the 15 current study was to predict the contribution of each site. The hydrolysis-time profiles of TU 16 incubation in human liver microsomes and Caco-2 cell homogenate were used to predict hepatic 17 and intestinal first-pass metabolism, respectively. The in vitro half-life (t1/2 inv) for the hydrolysis of 18 TU in microsomal mixtures was 28.31 ± 3.51 min. By applying the "well-stirred" model, the fraction 19 of TU that could escape hepatic first-pass metabolism (FH) was predicted as 0.915 ± 0.009. The 20 incubation of TU in Caco-2 cell homogenate yielded t1/2 inv of 109.28 ± 21.42 min which was applied 21 in "Q gut" model to estimate the fraction of TU that would escape intestinal first-pass metabolism 22 (FG) as 0.114 ± 0.02. Accordingly, only 11% of the absorbed fraction of TU could escape intestinal 23 metabolism while 91% of which can pass hepatic metabolism. Hence, compared to the liver, the 24 intestinal wall is the main site where TU is significantly metabolised during first-pass effect. 2

Application of biorelevant saliva-based dissolution for optimisation of orally disintegrating formulations of felodipine

The oral cavity is of great importance to the performance of orally retained formulations, including: orally disintegrating tablets, taste-masked formulations, and buccal/sublingual delivery systems. With regards to in vitro dissolution assessment of these dosage forms, human saliva should be represented by the dissolution media. Currently there is no general consensus regarding oral cavity dissolution. In this study pooled human saliva was characterised and utilised as dissolution media for biorelevant oral cavity dissolution studies and to assess drug release. Lipophilic drug felodipine with challenging biopharmaceutical properties was selected for assessment in oral cavity dissolution studies. These saliva dissolution studies investigated for the first time how biorelevant dissolution can be implemented as a screening tool to guide the formulation development process and to predict dosage form performance within the mouth. In this study a combination of three dissolution enhancement strategies (cryomilling, solid dispersion, and inclusion complexation) were employed to eventually increase the concentration of felodipine in saliva 150-fold. Using this successful formulation strategy orally disintegrating tablets of felodipine were produced. Interestingly, the percentage release of felodipine in compendial dissolution apparatus was shown to be over 80% after 10 minutes. On the other hand, salivabased dissolution showed that percentage release of felodipine was only 0.2% after 10 minutes using the same formulation. This discrepancy in drug release between dissolution media highlights the need for biorelevant dissolution apparatus for the oral cavity to reliably assess performance of relevant dosage forms in vitro

Oral administration of cannabis with lipids leads to high levels of cannabinoids in the intestinal lymphatic system and prominent immunomodulation

Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) have well documented immunomodulatory effects in vitro, but not following oral administration in humans. Here we show that oral co-administration of cannabinoids with lipids can substantially increase their intestinal lymphatic transport in rats. CBD concentrations in the lymph were 250-fold higher than in plasma, while THC concentrations in the lymph were 100-fold higher than in plasma. Since cannabinoids are currently in clinical use for the treatment of spasticity in multiple sclerosis (MS) patients and to alleviate nausea and vomiting associated with chemotherapy in cancer patients, lymphocytes from those patients were used to assess the immunomodulatory effects of cannabinoids. The levels of cannabinoids recovered in the intestinal lymphatic system, but not in plasma, were substantially above the immunomodulatory threshold in murine and human lymphocytes. CBD showed higher immunosuppressive effects than THC. Moreover, immune cells from MS patients were more susceptible to the immunosuppressive effects of cannabinoids than those from healthy volunteers or cancer patients. Therefore, administering cannabinoids with a high-fat meal or in lipid-based formulations has the potential to be a therapeutic approach to improve the treatment of MS, or indeed other autoimmune disorders. However, intestinal lymphatic transport of cannabinoids in immunocompromised patients requires caution

Quantitative analysis of lab-to-lab variability in Caco-2 permeability assays

In this study, Caco-2 permeability results from different laboratories were compared. Six different sets of apparent permeability coefficient (Papp) values reported in the literature were compared to experimental Papp obtained in our laboratory. The differences were assessed by determining the root mean square error (RMSE) values between the datasets, which reached levels as high as 0.581 for the training set compounds, i.e. ten compounds with known effective human permeability (Peff). The consequences of these differences in Papp for prediction of oral drug absorption were demonstrated by introducing the Papp into the absorption and pharmacokinetics simulation software application GastroPlus™ for prediction of the fraction absorbed (Fa) in humans using calibrated “user-defined permeability models”. The RMSE were calculated to assess the differences between the simulated Fa and experimental values reported in the literature. The RMSE for Fa simulated with the permeability model calibrated using experimental Papp from our laboratory was 0.128. When the calibration was performed using Papp from literature datasets, the RMSE values for Fa were higher in all cases except one. This study shows quantitative lab-to-lab variability of Caco-2 permeability results and the potential consequences this can have in the use of these results for predicting intestinal absorption of drugs

Dietary fats and pharmaceutical lipid excipients increase systemic exposure to orally administered cannabis and cannabis-based medicines

There has been an escalating interest in the medicinal use of Cannabis sativa in recent years. Cannabis is often administered orally with fat-containing foods, or in lipid-based pharmaceutical preparations. However, the impact of lipids on the exposure of patients to cannabis components has not been explored. Therefore, the aim of this study is to elucidate the effect of oral co-administration of lipids on the exposure to two main active cannabinoids, ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD). In this study, oral co-administration of lipids enhanced the systemic exposure of rats to THC and CBD by 2.5-fold and 3-fold, respectively, compared to lipid-free formulations. In vitro lipolysis was conducted to explore the effect of lipids on the intestinal solubilisation of cannabinoids. More than 30% of THC and CBD were distributed into micellar fraction following lipolysis, suggesting that at least one-third of the administered dose will be available for absorption following co-administration with lipids. Both cannabinoids showed very high affinity for artificial CM-like particles, as well as for rat and human CM, suggesting high potential for intestinal lymphatic transport. Moreover, comparable affinity of cannabinoids for rat and human CM suggests that similar increased exposure effects may be expected in humans. In conclusion, co-administration of dietary lipids or pharmaceutical lipid excipients has the potential to substantially increase the exposure to orally administered cannabis and cannabis-based medicines. The increase in patient exposure to cannabinoids is of high clinical importance as it could affect the therapeutic effect, but also toxicity, of orally administered cannabis or cannabis-based medicines

Natural sesame oil is superior to pre-digested lipid formulations and purified triglycerides in promoting the intestinal lymphatic transport and systemic bioavailability of cannabidiol

© 2021 Elsevier B.V. Lipid-based formulations play a significant role in oral delivery of lipophilic drugs. Previous studies have shown that natural sesame oil promotes the intestinal lymphatic transport and oral bioavailability of the highly lipophilic drug cannabidiol (CBD). However, both lymphatic transport and systemic bioavailability were also associated with considerable variability. The aim of this study was to test the hypothesis that pre-digested lipid formulations (oleic acid, linoleic acid, oleic acid with 2-oleoylglycerol, oleic acid with 2-oleoylglycerol and oleic acid with glycerol) could reduce variability and increase the extent of the intestinal lymphatic transport and oral bioavailability of CBD. The in vivo studies in rats showed that pre-digested or purified triglyceride did not improve the lymphatic transport and bioavailability of CBD in comparison to sesame oil. Moreover, the results suggest that both the absorption of lipids and the absorption of co-administered CBD were more efficient following administration of natural sesame oil vehicle compared with pre-digested lipids or purified trioleate. Although multiple small molecule constituents and unique fatty acid compositions could potentially contribute to a better performance of sesame oil in oral absorption of lipids or CBD, further investigation will be needed to identify the mechanisms involved

Simple and sensitive HPLC-UV method for determination of bexarotene in rat plasma

Bexarotene is currently marketed for treatment of cutaneous T-cell lymphoma and there has been growing interest in its therapeutic effectiveness for other cancers. Neuroprotective effects of bexarotene have also been reported. In this study, a simple, sensitive and cost-efficient bioanalytical method for determination of bexarotene in rat plasma was developed and fully validated. The method utilises protein precipitation with acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (10:1, v/v). An HPLC-UV system with a Waters Atlantis C18 column and a mobile phase of acetonitrile-ammonium acetate buffer (10 mM, pH 4.1) at a ratio of 75:25 (v/v), flow rate 0.2 mL/min was used. Chromatograms were observed by a UV detector with wavelength set to 259 nm. Intra- and inter-day validations were performed and sample stability tests were conducted at various conditions. The applicability of the method was demonstrated by a pharmacokinetic study in rats. Intravenous bolus dose of 2.5 mg/kg was administered to rats and samples were obtained at predetermined time points. As a result, pharmacokinetic parameters of AUCinf (4668 ± 452 h ng/mL), C0 (6219 ± 1068 ng/mL) and t1/2 (1.15 ± 0.02 h) were obtained. In addition, the developed method was further applied to human and mouse plasma to assess the suitability of the method for samples from other species

A novel nucleoside rescue metabolic pathway may be responsible for therapeutic effect of orally administered cordycepin

Although adenosine and its analogues have been assessed in the past as potential drug candidates due to the important role of adenosine in physiology, only little is known about their absorption following oral administration. In this work, we have studied the oral absorption and disposition pathways of cordycepin, an adenosine analogue. In vitro biopharmaceutical properties and in vivo oral absorption and disposition of cordycepin were assessed in rats. Despite the fact that numerous studies showed efficacy following oral dosing of cordycepin, we found that intact cordycepin was not absorbed following oral administration to rats. However, 3′-deoxyinosine, a metabolite of cordycepin previously considered to be inactive, was absorbed into the systemic blood circulation. Further investigation was performed to study the conversion of 3′-deoxyinosine to cordycepin 5′-triphosphate in vitro using macrophage-like RAW264.7 cells. It demonstrated that cordycepin 5′-triphosphate, the active metabolite of cordycepin, can be formed not only from cordycepin, but also from 3′-deoxyinosine. The novel nucleoside rescue metabolic pathway proposed in this study could be responsible for therapeutic effects of adenosine and other analogues of adenosine following oral administration. These findings may have importance in understanding the physiology and pathophysiology associated with adenosine, as well as drug discovery and development utilising adenosine analogues