Tomi Järvinen

Cyclodextrins in drug delivery

Cyclodextrins are a family of cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity. Cyclodextrin molecules are relatively large with a number of hydrogen donors and acceptors and, thus, in general they do not permeate lipophilic membranes. In the pharmaceutical industry cyclodextrins have mainly been used as complexing agents to increase aqueous solubility of poorly soluble drugs, and to increase their bioavailability and stability. Studies in both humans and animals have shown that cyclodextrins can be used to improve drug delivery from almost any type of drug formulation. However, the addition of cyclodextrins to existing formulations without further optimisation will seldom result in acceptable outcome. Currently there are ~ 30 different pharmaceutical products worldwide containing drug/cyclodextrin complexes on the market.

Ocular absorption following topical delivery

Abstract Most ocular diseases are treated with topical application of eyedrops. After instillation of an eyedrop, typically less than 5% of the applied drug penetrates the cornea and reaches intraocular tissues, while a major fraction of the instilled dose is absorbed and enters the systemic circulation. Ocular absorption of topically applied ophthalmic drugs is limited by rapid precorneal drug elimination due to solution drainage and systemic absorption from the conjunctival sac. Another factor that limits ocular absorption is the corneal epithelial barrier. In the eye drugs are distributed from the aqueous humor to intraocular tissues and eliminated mainly via aqueous humor turnover and venous blood flow in the anterior uvea. Because of the publication of several reviews [1–3] on ocular drug absorption this review will focus on the most recent literature.

Despite substantial degradation, 2‐arachidonoylglycerol is a potent full efficacy agonist mediating CB1 receptor‐dependent G‐protein activation in rat cerebellar membranes

Two endocannabinoids, arachidonoyl ethanolamide (AEA) and 2‐arachidonoylglycerol (2‐AG) bind and activate G‐protein‐coupled cannabinoid receptors, but limited data exist on their relative ability to activate G‐proteins. Here we assess agonist potency and efficacy of various cannabinoids, including 2‐AG, HU‐310 (2‐arachidonoyl glyceryl ether, a third putative endocannabinoid), HU‐313 (another ether analogue of 2‐AG), AEA, R‐methanandamide (an enzymatically stable analogue of AEA), and CP‐55,940 at rat brain CB1 receptors using agonist‐stimulated [35S]‐GTPγS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC. To enhance efficacy differences, agonist dose‐response curves were generated using increasing GDP concentrations. At 10−6u2003M GDP, all compounds, except HU‐313, produced full agonists responses ∼2.5 fold over basal. The superior efficacy of 2‐AG over all other compounds became evident by increasing GDP (10−5 and 10−4u2003M). In membrane incubations, 2‐AG was degraded by 85% whereas AEA and HU‐310 were stable. Pretreatment of membranes with phenylmethylsulphonyl fluoride inhibited 2‐AG degradation, resulting in 2 fold increase in agonist potency. Such pretreatment had no effect on AEA potency. Responses in brain sections were otherwise consistent with membrane binding data, but 2‐AG evoked only a weak signal in brain sections, apparently due to more extensive degradation. These data establish that even under conditions of substantial degradation, 2‐AG is a full efficacy agonist, clearly more potent than AEA, in mediating CB1 receptor‐dependent G‐protein activity in native membranes.

Evaluation of cytotoxicity of various ophthalmic drugs, eye drop excipients and cyclodextrins in an immortalized human corneal epithelial cell line

AbstractPurpose. An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/ toxicity by pharmaceuticals.nMethods. Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitrocytotoxicity tests, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay.nResults. Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine ≈ dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA2EDTA) > poly-vinyl alcohol (PVA) > methylparaben; and for CDs: α-CD > dimethyl-β-cyclodextrin (DM-β-CD) > sulfobutyl ether (β-cyclodextrin ((SBE)7m-β-CD) ≈ hydroxypropyl-β-cyclodextrin (HP-β-CD) > γ-CD. In consideration of the in vivoclinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure times (30 and 60 min).nConclusions. Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivocorneal toxicity of ocularly applied compounds.

Ophthalmic arachidonylethanolamide decreases intraocular pressure in normotensive rabbits

Arachidonylethanolamide (AEA) was the first anandamide to be identified as an endogenous ligand for the cannabinoid receptor of porcine brain. Since cannabinoids have shown some value in the reduction of ocular hypertension, the title compound was evaluated in normotensive rabbits as a possible topically applied agent for reducing intraocular pressure. AEA was dissolved in an aqueous solution of 2-hydroxy-propyl-beta-cyclodextrin. Single eyedrops (25 microliters) containing 3.13, 6.25, 31.25, 62.5 or 125.0 micrograms of AEA were instilled unilaterally into eyes of normotensive albino and pigmented rabbits. The intraocular pressures (IOPs) of these rabbits were then measured at fixed time intervals. The effect of AEA on IOP in treated and untreated (contralateral) eyes was similar in both types of rabbits. Administration of 31.25 micrograms of AEA caused an immediate IOP reduction in the treated eyes. AEA doses of 62.5 micrograms caused an initial increase and subsequent decrease of IOP in the treated eyes. In the untreated eyes, a marginal ocular hypotensive response of limited duration occurred immediately after administration of AEA at doses 31.25 or 62.5 micrograms. A significant increase (without subsequent decrease below baseline) in IOP occurred in treated eyes after a dose of 125.0 micrograms. The lowest dose (3.13 micrograms) did not have an effect on IOP. This study constitutes the first published demonstration that topical, unilateral administration of AEA significantly decreases IOP in normotensive albino and pigmented rabbits. Although the mechanism of action by which this compound produces its hypotensive effect in the eye is not known, the results suggest that AEA may prove useful in the investigation of glaucoma therapy.

Effects of topical anandamides on intraocular pressure in normotensive rabbits

A series of anandamide-type compounds were synthesized and studied for their effect on the intraocular pressure (IOP) of normotensive pigmented rabbits. Each test compound was dissolved in an aqueous 2-hydroxypropyl-beta-cyclodextrin solution and administered (31.25 - 62.5 micrograms) unilaterally to the eye. The most promising anandamides caused a statistically significant reduction of IOP in treated eyes, usually preceded by an initial transient elevation of IOP, compared to saline controls. In the contralateral untreated eyes, only a marginal or short hypotensive response was observed. Indomethacin pre-treatment (12.5 mg, s.c.) eliminated the IOP response to administered anandamides and arachidonic acid.

Identification of WIN55212-3 as a competitive neutral antagonist of the human cannabinoid CB2 receptor

1 Several G protein‐coupled receptors (GPCRs), including cannabinoid CB1 and CB2 receptors, show constitutive activity under heterologous expression. Such a tonic response is generated in the absence of an activating ligand, and can be inhibited by inverse agonists. Neutral antagonists, however, are silent at such receptors, but can reverse both agonist and inverse agonist responses. To date, no neutral antagonist for the CB2 receptor has been reported. 2 Here, by monitoring receptor‐dependent G protein activation, we demonstrate that WIN55212‐3 acts as a neutral antagonist at the human CB2 (hCB2) receptor. WIN55212‐3 alone, at concentrations 10−4u2003M, behaved as a silent ligand exhibiting no agonist or inverse agonist activity. However, WIN55212‐3 competitively antagonized cannabinoid agonist CP‐55,940‐stimulated responses (pA2 6.1). Importantly, the inverse agonism evoked by SR144528 in hCB2 was dose‐dependently reversed by WIN55212‐3 (pEC50 5.3±0.2), indicating true neutral antagonist behavior. 3 Furthermore, WIN55212‐3 also antagonized CB1 receptor signaling in a competitive manner (pA2 5.6), but behaved as a partial inverse agonist (pIC50 5.5±0.1) at the constitutively active human CB1. 4 Additionally, WIN55212‐3 antagonized signaling of the human melatonin MT1 receptor, with modest activity at the human muscarinic M4 receptor, but it was inactive towards several other GPCRs. 5 These data identify WIN55212‐3 as a true neutral hCB2 receptor antagonist. WIN55212‐3 offers a valuable tool for further characterization of ligand activities at the CB2 receptor and may serve as a lead compound in further efforts to develop more potent and selective neutral CB2 receptor antagonists.

Hydroxypropyl-β-cyclodextrin and its combination with hydroxypropyl-methylcellulose increases aqueous solubility of Δ9-tetrahydrocannabinol

Abstract Δ9-tetrahydrocannabinol (THC) is the main psychoactive constituent of Cannabis sativa L. and its therapeutic effects are currently under intensive study. However, THC has a very low aqueous solubility (1–2 ( μg mL ), which restricts its use as a pharmaceutical. The present study demonstrates that THC forms a drug-cyclodextrin complex in an aqueous solution of hydroxypropyl-β-cyclodextrin (HP-β-CD), resulting in a thousand-fold increase in THC solubility. This improvement in solubility can be further increased by adding 0.1% hydroxypropylmethylcellulose to the HP-β-CD solution. The present results suggest that the use of cyclodextrins might be a simple and useful method to overcome the poor water solubility of THC.

Effects of topical alpha-substituted anandamides on intraocular pressure in normotensive rabbits

AbstractPurpose. Anandamides have been observed to lower intraocular pressure in the rabbit eye, preceded by a period of hypertension. Amidases are thought to catabolize these compounds into their component parts, including arachidonic acid. Direct application of arachidonic acid has been observed to cause a marked rise of intraocular pressure. Thus, anandamide analogs resistant to catabolism were thought possibly devoid of this initial hypertension, and their effects on rabbit IOP investigated.nMethods. A series of chiral alpha-substituted anandamides were synthesized and studied for their effect on the intraocular pressure (IOP) of normotensive pigmented rabbits. Each test compound was dissolved in an aqueous 2-hydroxypropyl-β-cyclodextrin solution (containing 3% polyvinyl alcohol) and administered (62.5 μg) unilaterally to the eye.nResults. The most promising compounds caused a statistically significant reduction of IOP (vs. vehicle) in the treated eyes. Of these, the R-alpha-isopropyl compound exhibited the best activity tested. Unlike the alpha-unsubstituted analogs previously studied, hypotensive effects were not preceded by an initial elevation of IOP and indomethacin pre-treatment (12.5 mg, s.c.) did not eliminate the IOP response, as demonstrated by administered R-alpha-isopropyl anandamide.nConclusions. Catabolism of alpha-unsubstituted anandamides may account for their observed intraocular hypertensive effects. The physiological mechanism by which alpha-substituted anandamides work apparently differs from that of the more easily metabolized alpha-unsubstituted compounds.

Effects of Topical Anandamide-Transport Inhibitors, AM404 and Olvanil, on Intraocular Pressure in Normotensive Rabbits

AbstractPurpose. To evaluate the effects of topically applied anandamide transport inhibitors, AM404 and olvanil, on the intraocular pressure (IOP) of normotensive rabbits. To determine if the ocular hypotension induced by topical anandamide (AEA) can be potentiated by co-administered AM404.nMethods. Test compounds, in either hydroxypropyl-β-cyclodextrin (HP-β-CD) or propylene glycol, were administered unilaterally onto rabbit eyes. To determine if AM404 affects the IOP-profile of AEA, AM404 was administered ocularly 15 minutes before topical AEA. Phenylmethylsulfonyl fluoride (PMSF) (24 mg/kg, s.c.) was given 30 min before AEA to prevent its catabolism. IOPs of the treated and untreated eyes were measured. The cannabinoid agonist activities of AM404 and olvanil were studied by using [35S]GTPγS autoradiography.nResults. Topical AM404 (62.5 μg), in HP-β-CD vehicle, decreased IOP significantly in treated eyes. AM404 (62.5 μg) induced a significant IOP increase without subsequent decrease when given in propylene glycol vehicle. Olvanil (312.5 μg) caused a significant IOP reduction without provoking an initial hypertensive phase. These compounds did not significantly affect the IOP of untreated eyes. Co-administered AM404 (125 μg in HP-β-CD) had no significant effect on the IOP profile of AEA (62.5 μg).nConclusions. Ocular administration of AM404 or olvanil decreased IOP in rabbits, although AM404 can provoke an initial ocular hypertension and did not potentiate the IOP responses induced by exogenous AEA.