Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

Absorption Enhancing Effect of Cyclodextrins on Intranasally Administered Insulin in Rats

The absorption enhancing effect of -, -, and -cyclodextrin (CD), dimethyl--cyclodextrin (DMCD), and hydroxypropyl--cyclodextrin (HPCD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DMCD to the insulin solution resulted in a high bioavailability, 108.9 ± 36.4% (mean ± SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% -CD gave rise to an insulin bioavailability of 27.7 ± 11.5% (mean ± SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the -CD-containing solution as compared to the DMCD preparation. The other CDs, HPCD (5%), -CD (1.8%), and -CD (5%), did not have significant effects on nasal insulin absorption. DMCD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DMCD is a potent enhancer of nasal insulin absorption in rats.     

Preparation of Heptakis(2,6-di-O-ethyl)-β-cyclodextrin and Its Nuclear Magnetic Resonance Spectroscopic Characterization

Heptakis(2,6-di-O-ethyl)--cyclodextrin (DE--CyD) was prepared and its ¹H and ¹³C nuclear magnetic resonance (NMR) signals in DMSO-d₆ were unequivocally assigned by two-dimensional COSY and ROESY. The results on ¹H coupling constants indicated that all ethylated glucose units are in a ⁴C₁ chair conformation. The average spin-lattice relaxation times (T ₁) of ring carbons of DE--CyD were only slightly shorter, and their standard deviations from the mean T _l value were larger, than those of -cyclodextrin (-CyD) and heptakis(2,6-di-O-methyl)--cyclodextrin (DM--CyD), suggesting the presence of slightly irregular internal motion in the ethylated glucose units. The temperature dependence of chemical shift of DE--CyD in DMSO-d₆ suggested that the C3 hydroxyl protons may participate as proton donor in the intramolecular hydrogen bond to the C2 ethoxyl groups of neighboring glucose, and the intramolecular hydrogen bond of DE- and DM--CyDs is much stronger than that of -CyD, suggesting the stable macrocyclic ring structure of DE--CyD.     

The Interaction of Charged and Uncharged Drugs with Neutral (HP-β-CD) and Anionically Charged (SBE7-β-CD) β-Cyclodextrins

Purpose. The objective of this work was to determine the role that charge might play in the interaction of charged and uncharged drugs with neutral (2-hydroxypropyl--cyclodextrin, HP--CD) and anionically charged (SBE7--CD) modified -cyclodextrins. SBE7--CD is a sulfobutyl ether, sodium salt, derivative variably substituted on the 2-, 3- and the 6-positions of -cyclodextrin. The number seven refers to the average degree of substitution. Methods. The binding of the acidic drugs, indomethacin, naproxen and warfarin and the basic drugs, papaverine, thiabendazole, miconazole and cinnarizine with the two cyclodextrins was determined at 25°C as a function of pH and cyclodextrin concentration by the phase-solubility method. Results. Except for miconazole and cinnarizine (A_P-type diagrams), all other materials studied displayed A_L-type diagrams. By comparing the binding constants of both the charged and uncharged forms of the same drugs to both HP--CD and SBE7--CD, the following conclusions could be drawn. The binding constants for the neutral forms of the drugs were always greater with SBE7--CD than with HP--CD. For the anionic agents, the binding constants between SBE7--CD and HP--CD were similar while the binding constants for the cationic agents with SBE7--CD were superior to those of HP--CD, especially when compared with the neutral form of the same drug. Conclusions. A clear charge effect on complexation, attraction in the case of cationic drugs and perhaps inhibition in the case of anionic drugs, was seen with the SBE7--CD.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

Controlled Systemic Absorption and Increased Anesthetic Effect of Bupivacaine Following Epidural Administration of Bupivacaine-Hydroxypropyl-β-Cyclodextrin Complex

Purpose. To investigate the influence of complexation between bupivacaine and hydroxypropyl--cyclodextrin (HP--CD) on the systemic absorption and on the pharmacodynamic effect of bupivacaine following epidural administration in a rabbit model. Methods. Bupivacaine and bupivacaine-HP--CD complex were administered according to a randomized and cross-over design in six rabbits chronically instrumented with an epidural catheter. The plasma concentrations of bupivacaine and the duration and intensity of the motor blockade were evaluated. Results. Complexation with HP--CD led to a decrease in the maximum plasma concentration of bupivacaine. Individual absorption kinetics evaluated by Loo-Riegelman absorption analysis indicated that systemic absorption resulted from two parallel first-order processes. Only the faster absorption phase was slowed by complexation with HP--CD. The duration of the motor blockade was increased almost twice but the intensity was not modified. Conclusions. Complexation with HP--CD could be a promising drug delivery system to improve the therapeutic index of bupivacaine.     

Deformation Behaviors of Tolbutamide, Hydroxypropyl-β-Cyclodextrin, and Their Dispersions

Purpose. The deformation behaviors of compressedfreeze-dried and spray-dried tolbutamide/hydroxypropyl--cyclodextrinmolecular dispersions were evaluated and compared with similarly preparedtolbutamides (TBM), hydroxypropyl--cyclodextrins (HP--CD) and astheir physical dispersions. Methods. TBM, HP--CD, and their 1:1 moleculardispersions were prepared by freeze-drying and spray-drying, and physicaldispersions of TBM and HP--CD were blended. Deformation properties ofthe prepared materials were evaluated by using a compaction simulator andconstants derived from Heckel plots. Molecular dynamics (MD) simulationswere performed in order to gain a molecular-level view on the deformationbehavior of TBM-HP--CD inclusion complex. Results. The freeze-dried TBM polymorphic form II was lessprone to overall particle deformation than the spray-dried stable form I.Formation of molecular dispersions decreased the plastic and elasticbehaviors of these materials. Also, the MD simulations showed a reducedmolecular flexibility of the TBM-HP--CD inclusion complex, as comparedto HP--CD. Conclusions. The formation of TBM and HP--CDmolecular dispersion resulted in more rigid molecular arrangements, whichwere less prone to deformation than either HP--CDs or physicaldispersions. The results showed how differing molecular, solid, particle,and powder state properties affect the deformation properties of thematerials studied.     

Co-Administration of a Water-Soluble Polymer Increases the Usefulness of Cyclodextrins in Solid Oral Dosage Forms

Purpose. The aim of this study was to investigate the effect of cyclodextrins (-CD, HP--CD and (SBE)_7m--CD), and co-administration of a water-soluble polymer (HPMC) and cyclodextrins, on the oral bioavailability of glibenclamide in dogs. Methods. Effects of cyclodextrins on the aqueous solubility of glibenclamide, with and without hydroxypropylmethylcellulose (HPMC), were determined by a phase-solubility method. Solid inclusion complexes were prepared by freeze-drying. Glibenclamide was administered orally and intravenously to beagle dogs. Results. Aqueous solubility of glibenclamide increased as a function of cyclodextrin concentration, showing an A_L-type diagram for -CD and an A_p-type diagrams for both of the -CD derivatives studied. HPMC enhanced the solubilising effect of cyclodextrins, but did not affect the type of phase-solubility diagram. Orally administered glibenclamide and its physical mixture with HP--CD showed poor absolute bioavailability, while orally administered glibenclamide/cyclodextrin-complexes significantly enhanced the absolute bioavailability of glibenclamide. Orally administered glibenclamide/-CD/HPMC and glibenclamide/(SBE)_7m--CD/HPMC complexes showed similar absolute bioavailability compared to formulations not containing HPMC, even though 80% (in the case of (SBE)_7m--CD) or 40% (in the case of -CD) less cyclodextrin was used. Conclusions. The oral bioavailability of glibenclamide was significantly increased by cyclodextrin complexation. HPMC increased the solubilising effect of cyclodextrins and, therefore, the amount of cyclodextrin needed in the solid dosage form was significantly reduced by their co-administration. In conclusion, the pharmaceutical usefulness of cyclodextrins in oral administration may be substantially improved by co-administration of a water-soluble polymer.     

Design and Evaluation of an Osmotic Pump Tablet (OPT) for Chlorpromazine Using (SBE)7m-β-CD

Purpose. The purpose of this study was to develop a controlled-porosity osmotic pump tablet (OPT) which exhibits pH-independent release profiles for a basic drug using a sulfobutyl ether--cyclodextrin, (SBE)_7m--CD, which acts as both a solubilizer and as an osmotic agent. Methods. Chlorpromazine free base (CLP) was chosen as a model drug for this study. The release of CLP from osmotic pump tablets was studied in vitro. In vivo absorption of CLP from the OPT was evaluated in male beagle dogs. Results. The CLP release profile from an OPT prepared from a core tablet composed of a 1:10 molar ratio of CLP to (SBE)_7m--CD was pH-independent, and was controlled by modulating the membrane thickness of the OPT. Another cyclodextrin, hydroxypropyl--cyclodextrin (HP--CD), and a sugar mixture of lactose and fructose resulted in pH-dependent release at the same molar ratio. An in vivo absorption study in dogs with an OPT containing (SBE)_7m--CD correlated very well with the in vitro release profiles using the Japanese Pharmacopoeia dissolution method. Conclusions. In addition to serving as a solubilizer and osmotic agent, (SBE)_7m--CD can also serve as the controlling agent for pH independent release of CLP from OPTs. This system successfully modified the in vivo input rate of CLP without compromising oral bioavailability.     

Design and Evaluation of an Osmotic Pump Tablet (OPT) for Prednisolone,a Poorly Water Soluble Drug,Using (SBE)7m-β-CD

Purpose. The purpose of this study was to develop a controlled-porosity osmotic pump tablet (OPT) for poorly water soluble drugs using a sulfobutyl ether--cyclodextrin, (SBE)_7m--CD or Captisol, which acted as both a solubilizer and as an osmotic agent. Methods. Prednisolone (PDL) was chosen as a model drug for this study. The release of PDL from osmotic pump devices and tablets was studied. In vivo absorption of PDL from OPT was evaluated in male beagle dogs. Results. PDL release from the osmotic pump tablet with (SBE)_7m--CD was complete. Another cyclodextrin, hydroxypropyl--cyclodextrin (HP--CD), and a sugar mixture of lactose and fructose resulted in incomplete release. Although PDL release from the OPT with (SBE)_7m--CD and the sugar formulation displayed mainly zero-order release characteristics, the tablet utilizing HP--CD showed apparent first-order release characteristics. An in vivo absorption study in dogs correlated very well with the in vitro release profiles using the Japanese Pharmacopoeia dissolution method. Conclusions. The present results confirm that (SBE)_7m--CD can serve as both the solubilizer and the osmotic agent for OPT of PDL, and modify the input rate of PDL without compromising oral bioavailability.     

Protection Afforded by Maltosyl-β-cyclodextrin Against α-Chymotrypsin-Catalyzed Hydrolysis of a Luteinizing Hormone-Releasing Hormone Agonist, Buserelin Acetate

Purpose. The present study addresses how maltosyl--cyclodextrin (G₂--CyD) impacts upon the -chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G₂--CyD on the activity of the protease. Methods. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25°C and 37°C. The interaction of -chymotrypsin with G₂--CyD in the buffer solution was examined by differential scanning calorimetry. Results. G₂--CyD decelerated the -chymotrypsin-catalyzed hydrolysis of buserelin acetate to give the 1–3 tripeptide and the 4–9 hexapeptide fragments. This deceleration can be explained solely by a nonproductive encounter between a complex of the substrate with G₂--CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G₂--CyD on the proteolytic activity made a considerable contribution to the overall deceleration of the hydrolysis at higher CyD concentrations. Calorimetric studies indicate the presence of intermediate states in the thermal unfolding of -chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of -chymotrypsin was observed in the presence of G₂--CyD, suggesting reduced proteolytic activity upon binding to G₂--CyD. Conclusions. These results suggest that G₂--CyD at higher concentrations inhibits the proteolytic action of -chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.     

Formation of Fine Drug Particles by Cogrinding with Cyclodextrins. I. The Use of β-Cyclodextrin Anhydrate and Hydrate

Purpose. To improve the micromeritical properties of pranlukast (PRK) hydrate, a cogrinding process with cyclodextrin was used, and the formation of fine drug particles was investigated. Methods. PRK crystals were ground with either -cyclodextrin (-CD) anhydrate or -CD hydrate crystals at a mixing molar ratio of 2:1 (-CD:PRK) to prepare the ground mixtures (GMs). Powder X-ray diffraction measurement and particle size analysis were performed. Results. The two GMs differed from one another in appearance, wettability, and fine particle production. Quantitative determination demonstrated that when the -CD hydrate/PRK GM was dispersed in water, 96% of PRK loaded in GM became fine particles smaller than 0.8 m. In contrast, only 1.4% of PRK in GM transformed to fine particles in the case of -CD anhydrate/PRK GM. The PRK fine particles were considered to be dispersed as small crystals. The stability of PRK particles in the aqueous solution was improved by the addition of a water-soluble polymer. Conclusion. Cogrinding with a -CD of higher water content can be an effective method to prepare fine drug particles at the submicron level.     

β-Adrenoceptor subtypes in sections of rat and guinea-pig kidney

Summary The present autoradiographical study examines the distribution of the two -adrenoceptor subtypes in sections of rat and guinea-pig kidney. The radioligand [¹²⁵Iodo]-(-)-cyanopindolol was used for the labelling of -adrenoceptors and the selective -adrenoceptor blocking agents ICI 89-406 (₁-antagonist) and ICI 118-551 (₂-antagonist) were utilized to differentiate both subclasses unequivocally. -Adrenoceptors in rat kidney were found to be almost exclusively ₁. They were located mainly on glomeruli and to a lesser extent on the straight part of the distal tubules and on the cortical portion of the collecting ducts. Some ₂-adrenoceptors were localized around the corticomedullary junction. Grain localization in the autoradiograms was absent in the inner medulla and papilla. Glomeruli and distal tubules of the guinea-pig kidney also possess only ₁-adrenoceptors, but, in contrast to the rat, extremely high concentrations of ₂-adrenoceptors were associated with the straight part of the proximal tubules in the cortex and possibly with the cortical portion of the collecting duct. Labelling was not detected on the proximal convoluted tubule in either species.     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

Metabolism,Distribution, and Transdermal Permeation of a Soft Corticosteroid,Loteprednol Etabonate

The soft corticosteroid, loteprednol etabonate (chloromethyl 17-ethoxycarbonyloxy-11-hydroxy-3-oxoandrosta-1,4-diene-17-carboxylate), I, was designed based on the inactive metabolite approach. Accordingly, I should be metabolized by hydrolysis to the corresponding inactive cortienic acid derivative, II. The in vitro and in vivo metabolism of I indeed yielded mainly this inactive metabolite, which is more hydrophilic and thus readily eliminated from the body. Relatively high levels of I were found in tissues after intravenous administration of the drug in rats. The permeability of I through hairless mouse skin was comparable to what has been found for related hard steroids, without significant metabolism taking place in the skin.     

Antitumor Agents. 125. New 4β-Benzoylamino Derivatives of 4′-O-Demethyl-4-desoxypodophyllotoxin and 4β-Benzoyl Derivatives of 4′-O-Demethylpodophyllotoxin as Potent Inhibitors of Human DNA Topoisomerase II

A series of 4-benzoylamino (5-17) derivatives of 4-O-demethyl-4-desoxypodophyllotoxin and 4-benzoyl (18-20) derivatives of 4-O-demethyl podophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 5-13 and 15-17 are more potent than etoposide in causing DNA breakage, while compounds 9, 10, 13, 14, 16, and 20 are more active than etoposide in their inhibition of the human DNA topoisomerase II. The order for the enzyme inhibitory activity of the derivatives of 4-O-demethyl-4-desoxypodophyllotoxin is 4-arylamino > 4-benzylamino > 4-benzoylamino.     

Enzyme kinetics of zearalenone biotransformation: pH and cofactor effects

The aim of the present study was to investigate the hepatic biotransformation of the mycotoxin zearalenone (ZEA) in vitro using subcellular fractions of pig livers. The dependencies of the enzymatic reactions involved on the enzyme velocity, on the cofactor and on pH were analysed in both the microsomal fraction and the post-mitochondrial cell fraction. Finally, the inhibitory effects of various endogenous substrates on the enzymes involved (3- and 3-hydroxysteroid dehydrogenase) were examined. Significant differences were observed between the individual subcellular fractions in terms of prevailing metabolites and absolute amounts of the metabolites produced. Moreover, this study also demonstrated that the reactions for both subcellular fractions of porcine liver are dependent on the cofactor, as -zearalenol (-ZOL) formation increased in the presence of NADPH, whereas -zearalenol (-ZOL) production only increased in the presence of NADH (P<0.001). The optimal pH for -ZOL production was pH 5.6 and that for -ZOL formation pH 7.4. Subsequent inhibition studies showed significant inhibitory effects for 5-androstanedione>androstanedione>pregnenolone on -ZOL formation, whereas -ZOL production was only inhibited by pregnenolone. Finally, the contributions of 3- and 3-hydroxysteroid dehydrogenase during the bioconversion of ZEA are discussed in the context of these experiments.     

Sequential Processing of Human ProIL-1β by Caspase-1 and Subsequent Folding Determined by a Combined In Vitro and In Silico Approach

Purpose. Interleukin-1 is a multifunctional cytokine produced by activated monocytes and macrophages that requires caspase-1 (IL-1 converting enzyme/ICE) to process the 31kDa inactive precursor protein to the biologically active 17kDa peptide. This activation event involves ICE cleavage at Asp²⁷ (site 1) and Asp¹¹⁶ (site 2). To address the sequential processing at ICE cut sites we combined in vitro analysis and molecular modeling to investigate the sequence of molecular events. Methods. Pulse chase labeling followed by immunoprecipitation of IL-1 in activated human monocyte lysates demonstrated sequential cutting by ICE at site 1 before site 2 in vitro. To corroborate these findings, we constructed a homology model of proIL-1 after the crystal structure of another ICE substrate, human 1-antitrypsin (23% sequence identity). Results. Comparative modeling revealed that site 1 on proIL-1 is accessible to ICE but site 2 is not. Molecular dynamics simulations following ICE cleavage at site 1 and removal of the 3kDa amino-terminal fragment, rendered site 2 accessible to ICE. Conclusions. The close agreement between the in vitro and modeled behavior of IL-1 support our contention that IL-1 may be structurally related to 1-antitrypsin and also predicts that proIL-1 requires sequential processing for activation. These findings may facilitate the development of novel pharmacological agents that control posttranslational proIL-1 modification, thereby preventing excessive production of this potent inflammatory cytokine.     

Differential inhibition of biphenyl hydroxylation in perfused rat liver

Summary A differential inhibition of biphenyl hydroxylation by -naphthoflavone and metyrapone was observed in isolated pefused rat liver. -Naphthoflavone inhibited 2- and 4-hydroxylation in livers from -naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in -naphthoflavone-stimulated livers.Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.     

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--cyclodextrin (HP--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]GTPS autoradiography. Results. Topical AM404 (62.5 g), in HP--CD vehicle, decreased IOP significantly in treated eyes. AM404 (62.5 g) induced a significant IOP increase without subsequent decrease when given in propylene glycol vehicle. Olvanil (312.5 g) 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 g in HP--CD) had no significant effect on the IOP profile of AEA (62.5 g). 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.