Methazolamide 1% in Cyclodextrin Solution Lowers IOP in Human Ocular Hypertension

Elıńborg Guðmundsdo´ttir,1 Einar Stefańsson,1 Gyða Bjarnado´ttir,1 Jo´hanna F. Sigurjońsdo´ttir,2 Guðruń Guðmundsdo´ttir,1 Mar Masson,2 and Thorsteinn Loftsson2

PURPOSE. To formulate aqueous eye drops containing methazolamide 1% in cyclodextrin solution and to evaluate their effect on intraocular pressure (IOP) in a double-blind randomized trial in humans. Methazolamide, a carbonic anhydrase inhibitor (CAI), has been used in oral doses in the treatment of glaucoma but hitherto has not been successfully formulated in eye drops. In this study the effects of methazolamide are compared with those of dorzolamide (Trusopt).

METHODS. Methazolamide 1% was formulated in a 2-hydroxypropyl-␤-cyclodextrin with hydroxypropyl methylcellulose in aqueous solution. Eight persons with ocular hypertension were treated with the methazolamide-cyclodextrin eye drops and eight persons with dorzolamide (Trusopt), both groups at dosages of three times a day for 1 week. IOP was measured before treatment was begun and on days 1, 3, and 8 at 9 AM (peak) and 3 PM (trough).

RESULTS. After 1 week of treatment, the peak IOP in the methazolamide group had decreased from 24.4 Ϯ 2.1 mm Hg (mean Ϯ SD) to 21.0 Ϯ 2.0 mm Hg, which is a 14% pressure decrease (P ϭ 0.006). In the dorzolamide group, the peak IOP decreased from 23.3 Ϯ 2.1 mm Hg to 17.2 Ϯ 3.1 mm Hg, which is a 26% pressure decrease (P Ͻ 0.001). On average, the IOP declined 3.4 Ϯ 1.8 mm Hg after methazolamide administration and 6.1 Ϯ 3.6 mm Hg after dorzolamide.

CONCLUSIONS. Through cyclodextrin complexation, it is possible to produce topically active methazolamide eye drops that lower IOP. This is the ﬁrst double-blind clinical trial that demonstrates the efﬁcacy of the classic CAIs in eye drop formulation. (Invest Ophthalmol Vis Sci. 2000;41: 3552–3554)

arbonic anhydrase inhibitors (CAIs) were invented dur- in the aqueous tear film and also somewhat lipid soluble to be ing the middle of the 20th century, and acetazolamide able to penetrate the lipophilic barriers of the cornea.6 Few Cand methazolamide were used as systemically adminis- drugs fulfill both criteria. However, cyclodextrin can serve as a tered glaucoma drugs for the latter half of the century. Numer- vehicle that carries a hydrophobic drug in aqueous solution ous attempts to formulate these drugs as eyedrops were un- and releases the lipophilic molecule to the biologic membrane successful, and it was thought by many that their formulation such as the cornea. Methazolamide is a good example of this 1,2 in eye drops was impossible. With the help of a cyclodex- drug delivery dilemma. The ocular bioavailability of methazol- trin-based drug delivery system we have successfully formu- amide can be improved with the aid of a cyclodextrin-based lated acetazolamide, methazolamide, and ethoxyzolamide in drug delivery vehicle. eye drops and tested these in experimental animals and in an 2-Hydroxypropyl-␤-cyclodextrin (HP␤CD) is a cyclic oligo- 3–5 open pilot study in humans. We now report a double-blind saccharide with a hydrophilic outer surface and a lipophilic randomized clinical trial comparing the effect of methazol- cavity in the center. It is capable of forming inclusion com- amide in a cyclodextrin eye drop formulation to dorzolamide plexes with many lipophilic drugs by taking up a drug mole- eye drops (Trusopt; Merck Inc., Whitehouse Station, NJ). cule, either the whole molecule or part of it, into the cavity. In Methazolamide is nearly insoluble in water and aqueous this way it is possible to form water-soluble drug–cyclodextrin tear fluid, and this has made formulation of methazolamide eye complexes with water-insoluble lipophilic drugs. No covalent drops impossible until now.3 A drug molecule in eye drops bounds are formed or broken during the complex formation, must be water soluble, at least to some degree, to be dissolved and in aqueous solutions the complexes are readily dissoci- ated.6,7 Adding hydroxypropyl methylcellulose (HPMC) to the From the Departments of 1Ophthalmology and 2Pharmacy, Uni- solution increases the stability of the drug–cyclodextrin com- versity of Iceland, Reykjavı´k. plexes.3,8 The larger stability constant results in increased Presented in part at the annual meeting of the Association for complexation of the drug, and thus a higher concentration of Research in Vision and Ophthalmology in Fort Lauderdale, Florida, on the drug can be dissolved in a given amount of cyclodextrin.3 May 12 1999. Submitted for publication December 6, 1999; revised May 19, In general, cyclodextrin molecules do not penetrate biologic 2000; accepted May 22, 2000. membranes but act as penetration enhancers by assuring high Commercial relationships policy: P (ES, TL); all others N. ES and concentration of dissolved drug at the membrane surface. TL hold a patent for the drug delivery technology used in this study. Cyclodextrins increase the aqueous solubility of lipophilic wa- Corresponding author: Einar Stefańsson, Department of Ophthal- mology, University of Iceland, Landspı´tali, 101 Reykjavı´k, Iceland. ter-insoluble drugs without decreasing the drug molecules’ [email protected] ability to penetrate lipophilic biologic membranes.

Downloaded from iovs.arvojournals.org on 09/30/2021 IOVS, October 2000, Vol. 41, No. 11 Methazolamide in Ocular Hypertension 3553

TABLE 1. Intraocular Pressure at Baseline and After Administration of Eye Drops

(8 ؍ Methazolamide (n (8 ؍ Dorzolamide (n

Change from Change from IOP Baseline P IOP Baseline P

Baseline day 0 9 AM, peak 23.3 Ϯ 2.1 24.4 Ϯ 2.1 3 PM, trough 23.2 Ϯ 2.4 22.7 Ϯ 2.3 Treatment day 1 9 AM, peak 17.5 Ϯ 2.9 25% P Ͻ 0.001 21.9 Ϯ 1.6 10% P ϭ 0.017 3 PM, trough 18.2 Ϯ 1.8 21% P Ͻ 0.001 20.9 Ϯ 2.9 8% P ϭ 0.187 Treatment day 3 9 AM, peak 17.1 Ϯ 2.0 27% P Ͻ 0.001 21.2 Ϯ 2.2 13% P ϭ 0.012 3 PM, trough 17.6 Ϯ 1.6 24% P Ͻ 0.001 21.1 Ϯ 3.9 7% P ϭ 0.313 After 1 week 9 AM, peak 17.2 Ϯ 3.1 26% P Ͻ 0.001 21.0 Ϯ 2.1 14% P ϭ 0.006 3 PM, trough 17.2 Ϯ 1.5 26% P Ͻ 0.001 20.6 Ϯ 3.8 9% P ϭ 0.196

Data are means Ϯ SD.

The purpose of this study was to formulate eye drops study days with the baseline value at 9 AM on the baseline day containing methazolamide 1% in cyclodextrin solution and to 0, and a similar comparison was made for the 3 PM measure- evaluate its effect on IOP in humans with ocular hypertension ments, which were compared with the 3 PM measurement on and to compare it with dorzolamide in a double-blind random- day 0. ized clinical trial. The IOP was measured by Goldmann applanation tonom- etry two times at each measurement point, and the mean value recorded. Possible toxic effects were monitored throughout MATERIALS AND METHODS the study with evaluation of symptoms, best corrected Snellen visual acuity, and slit lamp examinations at all time points on study The institutional review board of Landspitali University Hospi- days. Student’s t-test was used for statistical analysis of the data. tal and the State Committee on Pharmaceutics in Reykjavı´k approved the protocol, which conformed to the tenets of the Declaration of Helsinki. Methazolamide eye drops were formu- RESULTS lated in an aqueous solution of HP␤CD with HPMC. One gram of methazolamide was added to 100 ml of an aqueous solution Table 1 shows the IOP data. Data are expressed as means Ϯ SD. containing 23 g HP␤CD (Encapsin; Janssen Biotech, Beerse, After 1 week of treatment, the peak IOP in the methazolamide Belgium), 0.1 g HPMC, 0.01 g benzalkonium chloride, and group had decreased from 24.4 Ϯ 2.1 to 21.0 Ϯ 2.1 mm Hg, 0.05 g EDTA. The solution was heated in an autoclave at 121°C which is a 14% pressure decrease (P ϭ 0.006). In the dorzol- for 40 minutes to promote the complexation between HP␤CD amide group, the peak IOP decreased from 23.3 Ϯ 2.1 to and methazolamide.3,8,9 The resultant solution was filtered 17.2 Ϯ 3.1 mm Hg, which is a 26% pressure decrease (P Ͻ through a 0.45-␮m nylon membrane and aseptically divided 0.001). On average, the IOP declined 3.4 Ϯ 1.8 mm Hg after into eye drop vials. Finally, the eye drop vials and their con- methazolamide administration and 6.1 Ϯ 3.6 mm Hg after dorzol- tents were sterilized in an autoclave at 121°C for 20 minutes amide. The difference between the methazolamide and dorzol- Patients with ocular hypertension (IOP Ͼ21 mm Hg) were amide groups did not reach statistical significance (P ϭ 0.07). recruited. Inclusion criteria included no previous treatment to Figure 1 shows the effect of methazolamide and dor- lower ocular pressure and no concurrent ocular therapy other zolamide on peak IOP. Looking at the trough effect (IOP at 3 than the study drug. Informed consent was obtained from each PM, just before the administration of the second drop) the IOP subject. Sixteen persons were included, 5 men and 11 women. in the methazolamide group decreased from 22.7 Ϯ 2.3 to 20.6 Ϯ The trial was randomized and double blind. We chose the eye 3.8 mm Hg, which is a 9% pressure decrease (P ϭ 0.196), and in in each patient with the higher IOP for the study. If both eyes the dorzolamide group IOP decreased from 23.2 Ϯ 2.4 to 17.2 Ϯ had the same IOP, we chose the eye with the worse visual 1.5 mm Hg, which is a 26% pressure decrease (P Ͻ 0.001). acuity to be the study eye. Lowering of IOP also occurred in the other eye that received no Dorzolamide and methazolamide eye drops were placed treatment, with IOP lowering at 9 AM from 22.6 Ϯ 5.0 to 21.3 Ϯ in identical bottles and were numbered in sequence and ran- 4.3 mm Hg in the methazolamide group, which is a 6% pressure domized for containing either dorzolamide or methazolamide. decrease and in the dorzolamide group from 21.3 Ϯ 3.1 to 19.9 Ϯ Eight persons self-administered the methazolamide-cyclodex- 3.8 mm Hg, which also is a 6% pressure decrease. trin eye drops and eight persons dorzolamide (Trusopt) eye The methazolamide-cyclodextrin eye drops were well tol- drops three times a day in the study eye for 1 week, at 7 AM, erated, and slit lamp examination of anterior segment revealed 3 PM, and 11 PM. There were no treatments of the other eye. no adverse effects of the drug. All persons in the methazol- The baseline IOP was measured on day 0 before treatment amide group mentioned a mild burning sensation on instilla- began at 9 AM and at 3 PM. The pressure was then measured tion but no other symptoms. In the dorzolamide group, all but on days 1 and 3 and after 1 week after treatment at 9 AM, one person reported a mild burning sensation on instillation (peak: 2 hours after the first drop) and 3 PM (trough: just and one person experienced transient (1–2 minutes) problems before the second drop). We compared IOP at 9 AM on the of focusing after 1 week of treatment.

Downloaded from iovs.arvojournals.org on 09/30/2021 3554 Guðmundsdo´ttir et al. IOVS, October 2000, Vol. 41, No. 11

have different afﬁnity and ability to inhibit human CA isoenzymes II and IV.

IC50 is the concentration at which a drug inhibits 50% of

enzyme activity, therefore lower IC50 values indicate higher

drug inhibitory activity. Dorzolamide has an IC50 for CA II of 0.18 nM and 6.9 nM for CA IV, whereas methazolamide has an 11 IC50 for CA II of 8.1 nM and 80.3 nM for CA IV. This indicates that dorzolamide is a 45 times and 12 times more potent inhibitor of the CA isoenzymes II and IV, respectively, than methazolamide. If we take into account that 1% methazolamide was compared with 2% dorzolamide, the cyclodextrin drug delivery system would have had to be 90 times more effective in drug delivery than the Trusopt system to equal the effect on

CAI II. Acetazolamide has an IC50 value for CA II of 3.4 nM and 14.7 nM for CA IV.11 Thus, acetazolamide would be expected to be more effective than methazolamide but less effective than dorzolamide. In our study the mean IOP decrease 2 hours after FIGURE 1. Effects of methazolamide and dorzolamide eye drops on peak intraocular pressure. The IOP measurements at 9 AM on day 0 instillation of the drops was 14% in the methazolamide group make the baseline IOP. Data show the percentage decrease from and 26% in the dorzolamide group. In our earlier study in baseline in IOP measurements at 9 AM on days 1 and 3 and after 1 which acetazolamide was formulated in cyclodextrin solution, week. the mean IOP decrease 2 hours after instillation was 15.6%.4 If we combine data from these studies on methazolamide, dorzolamide, and acetazolamide we conclude that there seems to DISCUSSION be a correlation between the affinity of the CAIs for the isoenzymes and their IOP-lowering effect. By formulating 1% methazolamide in a vehicle consisting of Our study shows for the first time in a double-blind ran- HP␤CD and HPMC, it became possible for the first time to domized trial that one of the classic CAIs, methazolamide, is produce topically effective methazolamide eye drops and dem- effective in an eye drop formulation based on HP␤CD and onstrate their efficacy in a double-blind randomized trial in HPMC. This has not been possible with any other drug delivery humans. Methazolamide is nearly insoluble in water and aque- system, even though attempts have been made over several ous tear fluid. Through cyclodextrin complexation, it is possi- decades to formulate the classic CAIs into eyedrops. ble to enhance aqueous solubility of water insoluble drugs, such as methazolamide, without affecting the lipophilic prop- erties of the drug molecule.6 Thus cyclodextrins act as true References drug carriers by keeping the drug molecules in solution and 1. Maren T. The development of topical carbonic anhydrase inhibi- delivering them to the surface of the eye where they penetrate tors. J Glaucoma. 1995;4:49–62. 6 the eye. 2. Pfeiffer N. Dorzolamide: development and clinical application of a In a previous study in rabbits we studied the dose– topical carbonic anhydrase inhibitor. Surv Ophthalmol. 1997;42: response and time course for methazolamide in cyclodextrin 137–151. solution. Methazolamide 1% was found to be more effective 3. Friðriksdottir H, Loftsson T, Stefansson E. Formulation and testing than 0.5% or 0.7%. The 1% solution is the highest concentra- of methazolamide cyclodextrin eye drop solutions. J Control Re- lease. 1997;44:95–99. tion that is practical, because higher concentrations of metha- 4. Loftsson T, Stefansson E, Kristinsson JK, et al. Topically effective zolamide and cyclodextrin are hypertonic. It showed a peak Acetazolamide eye-drop solution in man. Pharm Sci. 1996;2:277– effect 2 hours after administration, and the effect was dimin- 279. ished at 6 hours.3 On the basis of this study it was thought that 5. Loftsson T, Friðriksdo´ttir H, Stefańsson E, Tho´risdo´ttir S, Gud- application three times a day was appropriate. At this dose, the mundsson O¨ , Sigtho´rsson T. Topically effective ocular hypotensive frequency of administration is comparable to dorzolamide. In a acetazolamide and ethoxyzolamide formulations in rabbits. J Pharm Pharmacol. 1994;46:503–504. previous study in which we used cyclodextrin in aqueous tear 6. Loftsson T, Stefansson E. Effects of cyclodextrins on topical drug substitute in humans we established that cyclodextrin without delivery to the eye. Drug Dev Ind Pharm. 1997;23:473–481. 10 methazolamide has no effect on IOP. 7. Loftsson T, Friðriksdottir H, Thorisdottir S, et al. 2-Hydroxypropyl- The methazolamide-cyclodextrin eye drops were appar- ␤-cyclodextrin in topical carbonic anhydrase inhibitor formula- ently less effective than dorzolamide eye drops (Trusopt). The tions. Eur J Pharm Sci. 1994;1:175–180. most likely explanation for this difference is the difference in 8. Loftsson T, Stefansson E, Friðriksdottir H, Kristinsson JK. Effects of cyclodextrins and polymers on topical drug delivery to the eye- affinity of these drugs for the carbonic anhydrase (CA) isoen- evaluation in humans. Proc Int Symp Control Rel Bioact Mater. zymes. Seven isoenzymes of CA are known. In humans there 1996;23:453–454. are at least four isoenzymes of CA, and their concentrations 9. Loftsson T, Ja¨rvinen T. Cyclodextrins in ophthalmic drug delivery. vary in different organs.2 The ciliary body is rich in CA II, and Adv Drug Del Rev. 1999;36:59–79. CA IV is also present in the ciliary body.2 CA II is found in the 10. Guðmundsson OG, Tho´risdo´ttir S, Loftsson T, et al. 2-Hydroxypro- cellular cytoplasm, but CA IV is membrane bound. CA isoen- pyl-␤-cyclodextrin and cholesterol tear substitute [ARVO Ab- zyme II was traditionally viewed as the critical isoenzyme in stract]. Invest Ophthalmol Vis Sci. 1994;35(4): S1694. Abstract nr 2042. the formation af aqueous humor. This may still be the case, but 11. Sugrue M. The preclinical pharmacology of dorzolamide hydro- there is currently considerable interest in the possible involve- chloride, a topical carbonic anhydrase inhibitor. J Ocul Pharmacol ment of CA isoenzyme IV.11 Dorzolamide and methazolamide Ther. 1996;12:363–376.

Downloaded from iovs.arvojournals.org on 09/30/2021