Effect of ion pairing on in vitro transcorneal permeability of a Δ9-tetrahydrocannabinol prodrug: Potential in glaucoma therapy

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<ul><li><p>Effect of Ion Pairing on In Vitro Transcorneal Permeabilityof a 9-Tetrahydrocannabinol Prodrug: Potentialin Glaucoma Therapy</p><p>TUSHAR HINGORANI,1 WASEEM GUL,2,3,4 MAHMOUD ELSOHLY,2,3,4 MICHAEL A. REPKA,1,2,3 SOUMYAJIT MAJUMDAR1,2,3</p><p>1Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, Mississippi 38677</p><p>2Research Institute of Pharmaceutical Sciences, The University of Mississippi, University, Mississippi 38677</p><p>3National Center for Natural Product Research, The University of Mississippi, University, Mississippi 38677</p><p>4ElSohly Laboratories, Inc., Oxford, Mississippi 38655</p><p>Received 21 February 2011; revised 10 August 2011; accepted 27 September 2011</p><p>Published online 11 October 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22791</p><p>ABSTRACT: The aim of the present study was to evaluate and improve the in vitrotranscorneal permeability characteristics of 9-tetrahydrocannabinol (THC) through prodrugderivatization and formulation approaches. In vitro corneal permeability of THC and itshemisuccinate (THC-HS) and hemiglutarate (THC-HG) ester prodrugs and WIN 55-212-2(WIN), a synthetic cannabinoid, was determined using isolated rabbit cornea. The formula-tions studied included hydroxypropyl beta cyclodextrin (HP$CD) or randomly methylated betacyclodextrin (RM$CD), as well as prodrugion-pair complexes with L-arginine or tromethamine.Corneal permeability of WIN was found to be two-fold higher than THC in the presenceof HP$CD. THC-HS and THC-HG exhibited pH-dependent permeability. In the presence ofHP$CD, at pH 5 (donor solution pH), both prodrugs exhibited six-fold higher permeability com-pared with THC. However, permeability of the prodrugs was about three-fold lower than that ofTHC at pH 7.4. RM$CD, at pH 7.4, led to a significant improvement in permeability. Formationof ion-pair complexes markedly improved the solubility and permeability of THC-HG (seven-fold and threefold greater permeability compared with THC and WIN, respectively) at pH 7.4.The in vitro results demonstrate that the use of an ion-pair complex of THC-HG could be aneffective strategy for topical delivery of THC. 2011 Wiley Periodicals, Inc. and the AmericanPharmacists Association J Pharm Sci 101:616626, 2012Keywords: absorption; drug transport; formulation; passive diffusion/transport; permeability</p><p>INTRODUCTION</p><p>In 1971, Hepler and Frank1 published a report thatlinked marijuana smoking to a significant drop in</p><p>Abbreviations used: BCH, 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid; CB1, cannabinoid receptor type 1; CB2, cannabi-noid receptor type 2; DPBS, Dulbeccos phosphate buffered saline;HP$CD, hydroxypropyl beta cyclodextrin; IOP, intraocular pres-sure; IPBS, isotonic phosphate buffered saline; RM$CD, randomlymethylated beta cyclodextrin; THC, 9-tetrahydrocannabinol;THC-HS, 9-tetrahydrocannabinol hemisuccinate; THC-HG, 9-tetrahydrocannabinol hemiglutarate; THC-HGARG, ion pair of9-tetrahydrocannabinol hemiglutarate and L-arginine; THC-HGTRIS, ion pair of 9-tetrahydrocannabinol hemiglutarate andtromethamine; WIN, WIN 55-212-2.</p><p>Correspondence to: Soumyajit Majumdar (Telephone: +662-915-3793; Fax: +662-915-1177; E-mail: majumso@olemiss.edu)Journal of Pharmaceutical Sciences, Vol. 101, 616626 (2012) 2011 Wiley Periodicals, Inc. and the American Pharmacists Association</p><p>intraocular pressure (IOP). Because of its implica-tions in the treatment of glaucoma, this report stim-ulated intense research toward identification of theconstituents responsible for this pharmacological ac-tion. It was established that 9-tetrahydrocannabinol(THC, Fig. 1a), a primary active constituent of mar-ijuana, is one of the components responsible for theIOP lowering effects.2 During the course of further in-vestigations, a reduction in IOP was observed whenTHC was administered either orally or intravenouslybut not when applied topically.3,4 This lack of topicalactivity, although some reports did demonstrate thattopical delivery of THC significantly lowered IOP,5 ledresearchers to conclude that the IOP lowering mech-anism of THC was probably because of its centrallyacting hypotensive effect and not due to activation oflocal ocular receptors.</p><p>616 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 2, FEBRUARY 2012</p></li><li><p>ENHANCED TRANSCORNEAL PERMEATION OF THC PRODRUG 617</p><p>Figure 1. Chemical structures of (a) 9-tetrahydrocannabinol (THC), (B) 9-tetrahydrocannbinol hemisuccinate (THC-HS), (c) 9-tetrahydrocannbinol hemiglutarate(THC-HG), (d) WIN 55-212-2 (WIN).</p><p>However, recent studies suggest that THC canlower IOP and act as a neuroprotective agent bybinding to the cannabinoid receptors expressed inthe ocular tissues. In the 1990s, two cannabinoid re-ceptors type 1 and 2, CB1 and CB2, were identifiedand cloned.6 THC acts as an agonist for both CB1and CB2 receptors. Affinity values for the CB1 andCB2 receptors are 5.05 and 3.13 nM, respectively,7</p><p>whereas the half-maximal effective concentration val-ues for the CB1 and CB2 receptors are 6 and 0.4 nM,respectively.8 Although the distribution of cannabi-noid receptors in the body, since their identifica-tion, has been largely delineated, only recently havecannabinoid receptors been identified in the oculartissues. CB1 receptors are expressed in the trabec-ular meshwork, iris, ciliary body, and the retina,912</p><p>whereas CB2 receptors have been found in the retinaand trabecular meshwork.13 These locally expressedcannabinoid receptors are now believed to be involvedin the IOP lowering and neuroprotective activity of anumber of endocannabinoid and synthetic cannabi-noid derivatives.</p><p>There are two major pathways for the drainageof aqueous humor from the anterior ocular seg-ment: drainage through the Schlemms canal or theuveoscleral route. Activation of the CB1 receptorsin the ciliary muscle, by CB1 receptor agonists, in-duces contraction of the ciliary muscle.14 Contractionof the ciliary muscle leads to widening of the inter-cellular spaces in the trabecular meshwork and en-hances outflow of aqueous humor.15 Recently, bimato-prost, a prostaglandin analog that enhances uveoscle-ral outflow, has been shown to contract the humanciliary muscle through CB1-mediated mechanism.16</p><p>Furthermore, activation of CB1 receptors leads tofragmentation and reduction of actin stress fibersin the trabecular meshwork, further enhancing out-flow of aqueous humor.9 Thus, THC can reduce IOPthrough the local CB1/CB2 receptors. This can alsobe inferred from a previous clinical study. Merrittet al.5 demonstrated that 0.1% THC in mineral oilwhen given topically led to a 5.4 mm drop in IOP butwas accompanied with a 12 mm drop in systolic bloodpressure. A 10 mm drop in systolic blood pressure,</p><p>following systemic THC administration, should be as-sociated with less than a 1 mm drop in IOP.17 Also,0.05% THC in mineral oil, topically administered, ledto a 4.8 mm drop in IOP with no systemic hypotensiveeffect. Thus, these data suggest that topical THC isprobably acting locally through the ocular cannabi-noid receptors to reduce the IOP, and not through thesystemic pathway.</p><p>In glaucoma, a reduction in IOP is often not enoughto prevent or arrest the development or progressionof glaucoma-related optic neuropathy.18,19 Vision losscontinues even after significant IOP reduction hasbeen achieved. It has been suggested that neuroglialcell cytotoxicity in the optic nerve and retina leadsto visual field loss in glaucoma.20 Neuroglial cell tox-icity impairs macroglial glutamate metabolism andcauses microglia to release inflammatory cytokinesfollowing ischemia because of compression or vascu-lar occlusion. The released glutamate acts on recep-tors, including the N-methyl-D-aspartate subtype, onthe retinal ganglion cells to induce calcium influx andthe release of toxic reactive oxygen species, leading toapoptosis.20 Recent studies have demonstrated thatCB2 or nonspecific CB1/CB2 agonists were able toprotect retinal cells from oxidative stress but specificCB1 agonists had no effect.21 Intravitreally adminis-tered THC has been demonstrated to act as a neuro-protective in a rat model of glaucoma.20,22 Hampsonet al.23 also reported that the neuroprotective activ-ity of THC could also be independent of CB1 receptoractivation. Although the mechanism of neuroprotec-tion of THC is not yet clearly understood, it could bedue to the activation of CB2 receptors, its antioxidanteffect, or some other mechanism.2325 The current ev-idence clearly suggests that THC possesses both IOPlowering and neuroprotectant activity, which are in-dependent of each other.</p><p>Although significant efforts have been directed to-ward understanding the pharmacology of THC, de-sign of effective topical delivery strategies for THChas not seen much activity. In 1977, Green et al.26</p><p>published a paper comparing corneal penetration ofTHC from different oils and found that light mineraloil was the best of the four vehicles tested. The study</p><p>DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 2, FEBRUARY 2012</p></li><li><p>618 HINGORANI ET AL.</p><p>reported a 20% drop in IOP of normotensive rabbitswhen a 50:L dose (0.1%, w/w THC) was adminis-tered topically. However, plasma drug concentrationswere not reported. All further pharmacological stud-ies, with THC administered topically, were carried outusing light mineral oil as the vehicle. However, THCbeing a highly lipophilic molecule, with an aqueoussolubility of only 12 :g/mL and a log P of 6.3,27 itseffective partitioning from the oily vehicle into thetear film would be a suspect. Kearse and Green28 com-pared in vitro corneal permeability of THC from dif-ferent vehicles and observed that the permeability ofTHC from light mineral oil was only 1.86 108 cm/s.Thus, the lack of topical activity observed in the ear-lier in vivo reports could be due to the ineffective deliv-ery of THC to the target ocular tissues rather than ab-sence of local pharmacological activity. When higherdoses were administered, to increase the amount ofTHC permeating across the cornea, systemic side ef-fects were observed. Thus, development of a formu-lation that can effectively deliver THC across thecornea is needed prior to its evaluation for therapeuticactivity.</p><p>The aim of the current project was to improve theaqueous solubility and in vitro permeability of THCemploying complex formation and prodrug derivati-zation strategies. Dicarboxylic acid esters are com-monly used promoieties in prodrug derivatizationapproaches. These ester prodrugs exhibit higheraqueous solubility because they are ionized at phys-iological pH values. The THC hemisuccinate ester(THC-HS, Fig. 1b) and THC hemiglutarate ester(THC-HG, Fig. 1c) prodrugs were synthesized andevaluated for transcorneal permeability. The effect ofcyclodextrins and counterion adduct/complex forma-tion on the solubility and corneal permeability of THCand/or the two prodrugs were studied.</p><p>MATERIALS</p><p>Chemicals</p><p>Hydroxypropyl beta cyclodextrin (HP$CD), ran-domly methylated beta cyclodextrin (RM$CD),2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid(BCH), L-arginine (ARG), and Sigmacote R wereobtained from Sigma (St. Louis, Missouri). WIN-55-212-2 (WIN) was purchased from Tocris Bioscience(Ellisville, Missouri). All other chemicals were ob-tained from Fisher Scientific (St. Louis, Missouri).All solvents used for analysis were of high pressureliquid chromatography (HPLC) grade.</p><p>Animal Tissues</p><p>Recently, we have evaluated the effect of storageon corneas obtained from ocular globes preserved in</p><p>Hanks balanced salt solution and found the corneasto be equivalent to freshly excised corneas.29 Both ac-tive and passive transport processes in the preservedcorneas are intact for 24 h. Whole eye globes of al-bino New Zealand white rabbits were obtained fromPel Freez Biologicals (Rogers, Arkansas). Eyes wereshipped overnight in Hanks balanced salt solutionover wet ice and used immediately on receipt.</p><p>METHODS</p><p>Preparation of Dicarboxylic Acid Ester Prodrugs</p><p>Dicarboxylic acid prodrugs (THC-HS and THC-HG)were synthesized and characterized according to pre-viously published procedures.30</p><p>Solubility of the Prodrugs</p><p>Solubility in Buffers</p><p>As THC binds to plastic, all experiments were car-ried out in plastic vials/tubes coated with Sigmacote R</p><p>(Sigma) or borosilicate glass vials were used.31 Spe-cific measured quantities of stock solutions of THC-HS or THC-HG were transferred to the borosilicateglass or coated plastic vials and the organic sol-vent was evaporated using a stream of nitrogen gas.Dulbeccos phosphate-buffered saline (DPBS) or iso-tonic phosphate-buffered saline (IPBS) was thenadded to the vials and the resulting mixture was son-icated for 10 min to dislodge the drug sticking to con-tainer walls and allowed to equilibrate for 24 h at 25Cin a shaking water bath at 75 shakes per minute. Theresulting suspension was centrifuged at 16,000g ina Fisher Scientific acuSpin micro17R centrifuge for10 min using silicon-coated centrifuge tubes. The su-pernatant was collected and analyzed by HPLC.</p><p>Solubility in Cyclodextrins</p><p>Solubility of THC-HS and THC-HG in a 2.5% solu-tion of HP$CD or RM$CD in DPBS or IPBS was de-termined using methods described under solubility ofTHC-HS/THC-HG in buffer solutions.</p><p>Solubility in Presence of L-Arginine/Tromethamine</p><p>Formation of an ion-pair complex with a hydrophiliccounterion could lead to an improvement in the solu-bility of the drug. Aliquots of stock solutions of THC-HS and THC-HG were transferred to glass vials andthe organic solvent was evaporated using a streamof nitrogen gas. L-Arginine or tromethamine (TRIS)in IPBS was then added to specific concentrationsof the prodrug. The prodrug THC-HS/THC-HG andthe counterion ARG/TRIS were added in increasingconcentrations, keeping the ratio of drugcounterion</p><p>JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 2, FEBRUARY 2012 DOI 10.1002/jps</p></li><li><p>ENHANCED TRANSCORNEAL PERMEATION OF THC PRODRUG 619</p><p>constant (1:2). The combinations were then processedfor solubility determination following the same meth-ods as described under solubility of the prodrugs inbuffer solutions.</p><p>In Vitro Transcorneal Permeability Studies</p><p>Excess THC, THC-HS, THC-HG, and WIN were equi-librated in DPBS containing 2.5% HP$CD or RM$CD(pH adjusted to 5, 6, and 7.4) for 24 h at 25C ina shaking water bath. The supernatant containingthe drugcyclodextrin complex was used in the trans-port studies. Donor solutions were analyzed for drugcontent at the beginning and after completion of thein vitro permeability studies. Receiver solution for allpermeability studies with cyclodextrin formulationsconsisted of 2.5% HP$CD solution in DPBS with pHadjusted to 7.4.</p><p>Transcorneal permeability of the ion-pair com-plexes of THC-HG with L-arginine (THC-HGARG)and tromethamine (THC-HGTRIS) was also studied.THC-HG (1 mM) was equilibrated with ARG (2 mM) orTRIS (2 mM) in IPBS at 25C for 24 h in a shaking wa-ter bath. The supernatants were collected, analyzed,and used as the donor solution. For the studies in-vestigating permeability of the i...</p></li></ul>