Juha Juntunen

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol, and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.

In-vitro corneal permeation of cannabinoids and their water-soluble phosphate ester prodrugs.

Topically administered cannabinoids have been shown to reduce intraocular pressure by interacting with the ocular cannabinoid receptor. Most cannabinoids have very poor aqueous solubility, which limits their pharmaceutical development and usefulness. In this study, permeation of three cannabinoids (arachidonylethanolamide, R‐methanandamide and noladin ether) and their water‐soluble phosphate ester prodrugs across isolated rabbit cornea was investigated in vitro. Hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) was used to solubilize the parent cannabinoids in permeation studies to achieve the required concentration in donor and receiving cells. Highest fluxes were obtained with lipophilic parent compounds administered with HP‐β‐CD, and the fluxes of phosphate esters were 45–70% that of their corresponding parent compounds. Phosphate esters hydrolysed on the surface of the cornea or during the permeation to release the lipophilic parent compound, which further permeated the cornea. No phosphate esters were detected on the endothelial side of the cornea. Although the phosphate esters had lower fluxes than their corresponding parent compounds in these HP‐β‐CD formulations, the results are promising and the fluxes of phosphate esters are significantly higher than the fluxes of parent compounds administered as a suspension (due to their low aqueous solubility) without HP‐β‐CD.

A correlation of flux through a silicone membrane with flux through hairless mouse skin and human skin in vitro.

The maximum fluxes of 32 prodrugs and parabens through polydimethylsiloxane membranes from water (EXP log J(MPAQ)) have been correlated with the maximum flux of the same prodrugs and parabens through hairless mouse skin from water (EXP log J(MMAQ)): EXP log J(MMAQ)=0.608 EXP log J(MPAQ)-0.636, r(2)=0.743. The average of the absolute values for the differences between the EXP log J(MMAQ) and the log J(MMAQ) calculated from EXP log J(MPAQ) (Delta log J(MMAQ)) was 0.227 log units. Similarly the maximum fluxes of 11 unrelated permeants through human skin from water (EXP log J(MHAQ)) was correlated with the EXP log J(MPAQ) for the same permeants: EXP log J(MHAQ)=0.516 EXP log J(MPAQ)-0.922, r(2)=0.82 and Delta log J(MHAQ)=0.252 log units. Since the best fit of the databases for EXP log J(MPAQ), log J(MMAQ) and log J(MHAQ) was to the Roberts-Sloan (RS) model, and the dependency of RS on a balance in lipid and aqueous solubility for optimization of topical delivery has been established, the present correlation suggests that the flux through a silicone can be used to predict flux through mouse or human and that the physicochemical properties that lead to optimized flux through one membrane will lead to optimized flux through the others.

Anandamide prodrugs. 1. Water-soluble phosphate esters of arachidonylethanolamide and R-methanandamide.

Phosphate esters of arachidonylethanolamide (AEA) and R-methanandamide were synthesized and evaluated as water-soluble prodrugs. Various physicochemical properties (pK(a), partition coefficient, aqueous solubility) were determined for the synthesized phosphate esters. The chemical stability of phosphate esters was determined at pH 7.4. In vitro enzymatic hydrolysis rates were determined in 10% liver homogenate, and in a pure enzyme-containing (alkaline phosphatase) solution at pH 7.4. The intraocular pressure (IOP) lowering properties of R-methanandamide phosphate ester were tested on normotensive rabbits. The phosphate promoiety increased the aqueous solubility of the parent compounds by more than 16500-fold at pH 7.4. Phosphate esters were stable in buffer solutions, but rapidly hydrolyzed to their parent compounds in alkaline phosphatase solution (t(1/2)<<15 s) and liver homogenate (t(1/2)=8-9 min). The phosphate ester of R-methanandamide reduced IOP in rabbits. These results indicate that the phosphate esters of AEA and R-methanandamide are useful water-soluble prodrugs.

Modeling of flux through silicone membranes from water

Do the Roberts-Sloan (RS) or modified Kasting-Smith-Cooper (KSC) equations that provide good fit to data for maximum flux, from water through mouse or human skin also provide a good fit to data for maximum fluxes through silicone membranes (polydimethylsiloxane, PDMS). The maximum fluxes through silicone membranes from water (J(MPAQ)), molecular weights (MW), solubilities in isopropyl myristate (S(IPM)) and water (S(AQ)) of 31 prodrugs and one parent drug have been fitted to the RS equation, which includes a parameter for dependence on S(AQ), and the KSC equation, which does not, to determine which equation gave the better fit. In addition, the J(MPAQ), MW, S(AQ) and solubilities in octanol (S(OCT)) of 26 diverse molecules from other laboratories were collected and fitted to the RS and KSC equations to determine if the choice of lipid parameter (S(IPM) or S(OCT)) had an effect on which equation gave the better fit. RS gave the better fit to the present prodrug database where: logJ(MPAQ)=-2.454+0.716 logS(IPM)+0.284 logS(AQ)+0.00208 MW, r(2)=0.77. RS also gave the better fit to the database from other laboratories where: logJ(MPAQ)=-2.046+0.667 logS(OCT)+0.333 logS(AQ)-0.00374 MW, r(2)=0.878 after four obvious outliers were removed to give n=22. Thus, data for J(MPAQ) can be fitted to the RS equation, which also provides the best fit to maximum flux from water through mouse or human skin and includes a dependence on S(AQ).

A comparison of the fit of flux through hairless mouse skin from water data to three model equations

Data for the delivery of total species containing parent drugs from water through hairless mouse skin by prodrugs, logJ(MMAQ), has been fitted to the Roberts-Sloan, RS, the Kasting-Smith-Cooper, KSC, and Magnusson-Anissimov-Cross-Roberts, MACR, equations. The RS model which contains a parameter for the dependence of flux on solubility in water, S(AQ), as well as solubility in the lipid isopropyl myristate, S(IPM), gave the best fit: logJ(MMAQ)=-2.30+0.575 logS(IPM)+0.425 logS(AQ)-0.0016MW, r(2)=0.903. The values for the coefficients to the parameters are quite similar to those obtained when the RS model was fit to flux of solutes from water through human skin, logJ(MHAQ). There was no trend in predicting the under or over-performance of prodrugs based on their fit to the RS model and whether they were more or less soluble than their parent drugs. There was an inverse dependence of logJ(MMAQ) on partition coefficients or permeability coefficients similar to that observed for logJ(MHAQ). The similarities in trends for results for logJ(MMAQ) and logJ(MHAQ) suggests that design directives obtained from mouse skin can be extended to design new prodrugs or select new drugs for delivery through human skin.