Effects of topical anandamide-transport inhibitors, AM404 and olvanil, on intraocular pressure in normotensive rabbits.

PURPOSE: 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. METHODS: Test compounds, in either hydroxypropyl-beta-cyclodextrin (HP-beta-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]GTPyS autoradiography. RESULTS: Topical AM404 (62.5 micirog), in HP-beta-CD vehicle, decreased IOP significantly in treated eyes. AM404 (62.5 microg) induced a significant IOP increase without subsequent decrease when given in propylene glycol vehicle. Olvanil (312.5 microg) 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 microg in HP-beta-CD) had no significant effect on the IOP profile of AEA (62.5 microg). CONCLUSIONS: 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.

Effect of the CB1 receptor antagonist, SR141716A, on cannabinoid-induced ocular hypotension in normotensive rabbits.

The present study attempts to indirectly determine if a neuronal cannabinoid (CB1) receptor mediates the intraocular pressure (IOP) reduction effects of arachidonoyl ethanolamide (AEA), its R-alpha-isopropyl analog, and the non-classical cannabinoid, CP-55,940. A series of these cannabinoids were dissolved in an aqueous 10-20% 2-hydroxypropyl-beta-cyclodextrin (2-HP-beta-CD) solution (containing 3% polyvinyl alcohol) and administered (25-62.5 microg) unilaterally to normotensive rabbit eyes. This was repeated on animals pre-treated with a subcutaneous injection (2.5 mg/kg) of the highly specific CB1 receptor antagonist, SR 141716A, dissolved in an aqueous 42% 2-HP-beta-CD solution. AEA, its R-alpha-isopropyl analog, and CP-55,940 reduced IOP upon topical application to a greater degree than was detected in the untreated eye. This reduction was eliminated for the latter two compounds by subcutaneous (s.c.) pretreatment of the rabbits with the CB1 receptor antagonist, but the IOP properties of AEA remained unchanged. SR 141716A administered alone (s.c.), elevated the IOP of both eyes. A CB1 receptor seems involved in the IOP reduction induced by either R-alpha-isopropyl anandamide or CP-55,940. However, AEA apparently functions through a different mechanism.

Hydroxypropyl-beta-cyclodextrin and its combination with hydroxypropyl-methylcellulose increases aqueous solubility of delta9-tetrahydrocannabinol.

Delta9-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 microg/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-beta-cyclodextrin (HP-beta-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-beta-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.

PURPOSE: 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. METHODS: 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-beta-cyclodextrin solution (containing 3% polyvinyl alcohol) and administered (62.5 micrograms) unilaterally to the eye. RESULTS: 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. CONCLUSIONS: 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.

Hydroxypropyl-beta-cyclodextrin increases aqueous solubility and stability of anandamide.

Anandamide (arachidonylethanolamide; AEA) is an endogenous ligand for the cannabinoid receptor and its pharmacological effects are under intensive study. However, AEA has a low aqueous solubility and stability which may restrict its use and may eventually endanger the reliability of the obtained results. In the present study, it was found that AEA forms inclusion complexes with cyclodextrins (CDs), resulting in greater aqueous solubility and stability of AEA as AEA/CD complex. Aqueous solubility increased 1 000 to 30 000-fold, depending on the type of CD (10% solution) used. The half-life of AEA in aqueous hydroxypropyl-beta-cyclodextrin solution (10%) at 50 degree C was 2.9 years. These results suggest that CD-technology will be a very useful method to overcome the solubility and stability problems of AEA.

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.

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.

Repair of articular cartilage defects using mesenchymal stem cells.

Degeneration of articular cartilage in osteoarthritis is a serious medical problem. We have isolated a population of cells from the connective tissue of mammals termed mesenchymal stem cells (MSCs) for their apparent unlimited growth potential and their ability to differentiate into several phenotypes of the mesodermal lineage, including cartilage and bone. These qualities make them ideal candidates for cartilage repair. We isolated MSCs from adult rabbit muscle and cultured them in vitro into porous polyglycolic acid polymer matrices. The matrices were implanted into 3-mm-diameter full thickness defects in rabbit knees with empty polymer matrices serving as the contralateral controls. The implants were harvested 6 and 12 weeks postop. At 6 weeks, the controls contained fibrocartilage while the experimentals seemed to contain undifferentiated cells. By 12 weeks postop, the controls contained limited fibrocartilage and extensive connective tissue, but no subchondral bone. In contrast, the implants containing MSCs had a surface layer of cartilage approximately the same thickness as normal articular cartilage and normal-appearing subchondral bone. There was good integration of the implant with the surrounding tissue. Implantation of MSCs into cartilage defects appears to effect repair of both the articular cartilage and subchondral bone. Studies are ongoing to further characterize the use of MSCs for cartilage repair.

Mood alterations in older adults following acute exercise.

Limited research indicates positive affective change following acute bouts of exercise, but whether this improved affect among younger subjects may be generalized to older individuals is not known. The present study, then, examined the effects of a single bout of physical activity among older participants. 16 trained women (Mage = 64.5 +/- 7.6 yr.) completed an abbreviated Profile of Mood States prior to and immediately following a 75-min. session of aerobic line dancing. A series of one-way analyses of variance with repeated measures were used to examine differences between pre- and posttest subscores on mood states. Significant decreases following exercise in scores on Tension, Depression, Fatigue, and Anger and a significant increase in scores on Vigor relative to preexercise (control) scores were found. Global mood was significantly improved after the exercise session. No significant difference was found between pre- and postexercise measures of Confusion. Previous findings of significant improvements in affect immediately after an acute bout of exercise may be generalized to older adults. Repetition with a nonexercised control group is desirable.

Biological efficacy of boronated low-density lipoprotein for boron neutron capture therapy as measured in cell culture.

Low-density lipoproteins (LDLs) are known to be internalized by the cell through receptor-mediated mechanisms. There is evidence that LDLs may be taken up avidly by tumor cells to provide cholesterol for the synthesis of cell membranes. Thus, the possibility exists that LDLs may provide an ideal vehicle for the transport of boron to tumor cells for boron neutron capture therapy. A boronated analogue of LDL has recently been synthesized for possible application in boron neutron capture therapy. The analogue was tested in cell culture for uptake and biological efficacy in the thermal neutron beam at the Brookhaven Medical Research Reactor. It was found that boron concentrations 10 times higher than that required in tumors for boron neutron capture therapy were easily obtained and that the amount of uptake was consistent with a receptor-mediated binding mechanism. The measured intracellular concentration of approximately 240 micrograms 10B/g cells is significantly higher than that obtained with any other boron compound previously evaluated for possible clinical application.

Stereoselective N-oxygenation of zimeldine and homozimeldine by the flavin-containing monooxygenase.

The metabolism of (Z)- and (E)-zimeldine and (Z)- and (E)-homozimeldine in hepatic rat and hog microsomes is described. The major metabolite observed in all cases examined was the tertiary amine N-oxide and it was formed at a rate 7-20 times that of norzimeldine or homonorzimeldine. N-Oxygenation requires NADPH and is stimulated by n-octylamine. Thiobenzamide and methimazole significantly inhibit N-oxide formation whereas heat pretreatment of microsomes completely abolishes N-oxide formation, strongly suggesting that zimeldine N-oxygenation if solely dependent on the flavin-containing monooxygenase. Hog liver microsomes N-oxygenate the Z-allylic and homoallylic tertiary amines in marked preference to the E-isomers, whereas rat liver microsomes N-oxygenate E-isomers to a greater extent than Z-isomers. Thus, opposite stereoselectivity for zimeldine N-oxygenation occurs in rat liver and hog liver microsomes.