Eye (1990) 4, 603-606

Penetration of Topically Applied Betamethasone Sodium Phosphate into Human Aqueous Humour

D. G. WATSON,* C. N. 1. McGHEE,*t 1. M. MIDGLEY,* G. N. DUTTON,t M. 1. NOBLEt Glasgow

Summary Gas Chromatography combined with Negative Chemical Ionisation Mass Spec­ trometry (GCMS) was used to determine the absorption of topically applied beta­ methasone sodium phosphate into the aqueous humour of human subjects undergoing routine intraocular surgery. The Betamethasone concentration was greatest in the interval 91-120 minutes following topical administration (mean peak

concentration = 7.7 ng/ml). At twelve hours post instillation the mean concentration of Betamethasone was 2.5 ng/ml and detectable levels were recorded in the aqueous humour 24 hours after application (mean concentration 0.4 ng/ml).

The authors have previously reported data on commonly used, commercially available, the penetration of dexamethasone alcohol topical corticosteroid preparations. 0.1%,1 prednisolone sodium phosphate 0.5%2 and prednisolone acetate 1.0%3 into Subjects and methods human aqueous humour utilising the highly Patients who were scheduled to undergo rou­ sensitive and specific technique of Gas Chro­ tine cataract surgery were recruited to the matography with Negative Chemical Ionisa­ study and informed consent was obtained in tion Mass Spectrometry (GCMS)." Limited all cases. Patients with corneal disease or data is available on the direct measurement of inflammatory ocular conditions which might Betamethasone in aqueous humour of animal affect steroid penetration into the eye were models' following topical application. We excluded from the study. None of the patients used GCMS to analyse the penetration of had been prescribed topical or systemic ste­ betamethasone sodium phosphate into the roids in the six months prior to surgery. Sixty­ aqueous humour at varying times following six eyes of 66 patients were included. The the topical application of eyedrops to human mean age of patients included was 72 years volunteer subjects undergoing routine catar­ (range 56-92 years). act extraction. This work forms part of a Fifty microlitres of betamethasone sodium larger ongoing study which aims to establish phosphate (0.1%) solution (Betnesol: Glaxo the aqueous humour pharmacokinetics of the Ltd, UK) were introduced by micropipette

* Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Strathc1yde, Royal College Buildings, 204 George Street, Glasgow GIIXW. t Department of Ophthalmology, Southern General Hospital, ' Govan Road, Glasgow. :j: Tennent Institute of Ophthalmology, University of Glasgow, Western Infirmary, Glasgow G 11 6NT.

Correspondence to: Dr C. N. J. McGhee, Senior Registrar, Tennent Institute of Ophthalmology, Western Infirmary, Glasgow G 11 6NT. 604 D. G. WATSON ET AL.

Table I Mean concentration (nglml) (± standard trideuterated compound added before extrac­ error of mean) of betamethasone in human aqueous tion. Thus the deuterated isotopomer of beta­ humour at various times (min) following the topical methasone co-chroma to graphed with the administration of betamethasone sodium phosphate corresponding undeuterated substance and 0.1%. yielded ions in its mass spectrum equivalent to

Time No. of Mean conc'n in those obtained from the undeuterated com­ interval eyes aqueous humour pound together with a mass increment of 2-3 (mins) (n=66) (ng/ml) ± (SEM) atomic mass units. This technique is suitable for the quantification of betamethasone in the 0-30 5 0.0 ± 0.0 0.1-10 ng range. 31--60 13 3.8 ± 1.0 61-90 9 4.2 ± 0.6 In order to detect if betamethasone sodium 91-120 10 7.7 ± 1.8 phospate was present in the aqueous humour 121-180 11 6.5 ± 1.5 ten samples were incubated with acid phos­ 181-240 2 6.9 ± 1.1 phatase following the initial extraction with 241-460 2 5.3 ± 2.1 ethyl acetate (acid phosphatase was found to 461-760 5 2.5 ± 0.6 761-1060 4 0.7 ± 0.5 be effective in releasing betamethasone from 1061-1430 5 0.4 ± 0.2 its phosphate in standard solutions). The enzymatically treated solution was then pro­ into the lower conjunctival fornix of the eye cessed as described above for the untreated aqueous humour. No additional betametha­ being prepared for surgery at varied times up sone could be detected after treatment with to 24 hours pre-operatively. Two drops each acid phosphatase. of Phenylephrine 10% and Cyclopentolate 1% were administered one hour prior to sur­ Results gery to obtain mydriasis. The concentrations of betamethasone in Tw enty-nine of the operations were per­ human aqueous humour at varied time inter­ formed under general anaesthesia and 37 vals following the topical administration of were performed under retrobulbar anaes­ Betamethasone Sodium Phosphate are shown thesia. 0.05 to 0.15 millilitres of aqueous in Table I. The concentrations of betametha­ humour were aspirated with a tuberculin sone in aqueous humour vs time from instill­ syringe through a 25 gauge needle after a par­ ation is shown in Figure 1. Peak mean 91- tial thickness corneoscleral incision had been concentrations were recorded between 120 made but before the anterior chamber had minutes after application. Levels which 30% been entered. The interval between the are approximately of the peak drug con­ instillation of betamethasone sodium phos­ centration were measured between eight and 12 24 phate and the aspiration of aqueous humour hours and trace levels were recorded at was noted. The aqueous samples were hours. immediately frozen and stored at -20°C until Concentration ng/ml analysed by GCMS.

Two nanograms of deuterated betametha­ 9 sone (containing largely the trideuterated 7 derivative) were added to the sample of aque­

ous humour (which had previously been 5 diluted to 1 ml with high purity water). The aqueous layer was extracted with ethyl ace­ 3 tate (2 xI ml) and the extract chemically treated to give the di-methoxime tris-trimeth­ 4 ylsilyl ether derivative. The derivatised o 200 400 600 800 1000 1200 extract was then subjected to GCMS in the Time (minutes) negative chemical ionisation mode; the Fig. 1. Mean peak concentrations of betamethaspne undeuterated corticosteroid present in the (nglml ± SEM) in human aqueous humour vs time aqueous humour was identified and quanti­ (mins) following topical application of betamethasone fiedwith reference to the known amount of dil sodium phosphate 0.1%. PENETRATION OF TOPICALLY APPLIED BETAMETHASONE SODIUM PHOSPHATE 605

There were no statistical differences in sitive technique. The remit of our study was to aqueous betamethasone concentrations when analyse the intraocular penetration of com­ the results from patients receiving general mercially available topical corticosteroids, anaesthetic were compared to those who had therefore, our data is limited to the 0.1% con­ undergone retrobulbar anaesthesia. centration of betamethasone sodium phos­ phate. None the less it is worth noting that Discussion previous animal studies have shown that beta­ In human aqueous humour the mean peak methasone sodium phosphate, prednisolone concentration of betamethasone (7.7 ng/ml) sodium phosphate, and prednisolone acetate is much lower than we have previously all achieve significantly higher aqueous levels 1.0% recorded for dexamethasone alcohol 0.1% when delivered in preparations when (31.0 ng/ml),1 prednisolone sodium phos­ compared to lower concentrations.5,11,12 phate 0.5% (25.6 ng/ml)2 or prednisolone ace­ Among the possible variables we found no tate 1.0% (669.6 ng/ml)3. However, the mean significant differences in aqueous levels of betamethasone when patients were grouped concentration of betamethasone does not fall by age, sex, or type of anaesthetic. It might be below 68% of the peak concentration within postulated that eyes with cataract do not eight hours of application, whereas, the peak behave identically to inflamed eyes, in which levels of the other three drugs fall to less than intraocular inflammation can interfere with 33% of peak concentration at eight hours. the epithelial barrier to a water soluble The lower mean peak concentration of beta­ corticosteroid,13 or that the application of methasone when compared to dexametha­ cyclopentolate and phenylephrine might sone and prednisolone acetate might be partly influencethe surface characteristics of the eye explained by its preparation as a phosphate; and therefore the penetration of topical ste­ such corticosteroid preparations have been roids. Unfortunately, these are variables shown to be polar and thus the intact corneal which are unavoidable in human research; the epithelium presents more of a barrier to their former we cannot influencethe latter we hope penetration than the more lipid soluble ace­ has been minimised by the standardisation of tate and alcohol corticosteroid derivatives.6,7,8 preoperative mydriatic doses. In such comparisons it is important to remem­ Although betame.thasone sodium phos­ ber that on a weight for weight basis dexame­ phate 0.1 % does not reach the mean peak thasone and betamethasone have a five to aqueous concentrations of dexamethasone seven fold greater systemic anti-inflammatory alcohol 0.1%, prednisolone sodium phos­ activity than prednisolone.9 However, it has phate 0.5% or prednisolone acetate 1.0% it been suggested on the basis of evidence from does have a longer aqueous half life. There­ animal models that these differences in fore topical application two to three times systemic anti-inflammatory activity may not daily may be sufficient to maintain peak beta­ 0 be directly extrapolated to the ocular milieu. 1 methasone concentrations in the aqueous To the authors' knowledge there are no pre­ humour. However, we have yet to analyse the viously published data on betamethasone additive effects of repeat application and as concentrations in human aqueous humour such the aqueous kinetics of the cortico­ following topical administration. However, steroids examined so far cannot be fully pre­ betamethasone has been studied in rabbit dicted. Hopefully when this data is available eyes following topical administration using more rational regimens may be applied for the radioimmunoassay techniques. 5 Using this topical administration of betamethasone and method peak aqueous humour concentrations the other topical ophthalmic corticosteroid of 72.2 ng/ml were recorded. It was also noted preparations. that greater aqueous humour concentrations The work described has been supported by a grant were obtained with 0.5% and 1.0% prep­ from the Scottish Hospital Endowments Research arations of betamet has one but at these higher Trust (Grant No. HERT 761). The authors are concentrations significant levels of beta­ grateful to Dr J. Williamson, Dr W. M. Doig, Dr P. methasone were detected in the contralateral M. Kyle and Dr J. Dudgeon for providing aqueous control eye perhaps suggesting an overly sen- samples. 606 D. G. WATSON ET AL.

Proprietary statement the bioavailability of ophthalmic steroids: None of the authors has any commercial or proprietary Development of an in vitro method of evaluation. interests in Betamethasone eyedrops or any other oph­ Arch Ophthalmol1984,102: 1808-9. thalmic corticosteroid eyedrops. 7 Kupferman A, Pratt MV, Suckewer K et al: Topi­ cally applied steroids in corneal disease: 11l. The References role of drug derivative in stromal absorption of 1 Watson DG, Noble Ml, Dutton GN et al: Penetra­ dexamethasone. Arch Ophthalmol 1974, 91: tion of topically applied dexamethasone alcohol 373-6. into human aqueous humour. Arch Ophthalmol 8 Hull DS, Hine lE, Edelhauser HF, Hyndiuk RA: 1988,106: 686-7. Permeability of the isolated rabbit cornea to corti­ 2 McGhee CN, Watson DG, Noble Ml et al: Pen­ costeriods. Invest Ophthalmol1974,13: 457-9. etration of topically applied prednisolone sodium Y Havener WH: Corticosteroid therapy in Ocular phosphate into human aqueous humour Eye Pharmacology (fifth edition) ed. William H. 1989,3: 463-7. Havener; CV Mosby Co, St Louis. 1983; 433-500. 3 McGhee CN, Watson DG, Midgley 1M et al: Pen­ 10 Leibowitz HM: Management of inflammation in the etration of Synthetic Corticosteroids into Human cornea and conjunctiva. Ophthalmology19 80; 87: Aqueous Humour. Eye19 90 (In press). 753-8. 4 Midgley 1M, Watson DG, Healey TM, Noble M: 11 Kupferman A and Leibowitz HM: Topically applied The quantification of synthetic corticosteroids steroids in corneal disease: VI. Kinetics of pred­ using isotope dilution gas chromatography nega­ nisolone sodium phosphate. Arch Opthalmol tive chemical ionisation mass spectrometry. Bio­ 1974,92: 331-4. med Environmental MassSpect 1988, 15: 479-83. 12 Leibowitz HM and Kupferman A: Kinetics of Topi: 5 Baba S, Mishima H, Miyachi Y: Levels of Cyclic­ cally administered prednisolone acetate. Arch AMP, Cyclic-GMP and betamethasone in the Ophthalmol1 976, 94: 1387-9. aqueous humour following topical administration 13 Cox WV, Kupferman A, Leibowitz HM: Topically of betamethasone in rabbit eyes. Hiroshima J applied steroids in corneal disease: 1. The role of Med Sci19 83,3: 301-4. inflammation in stromal absorption of dexame­ 6 Flint FR and Morton Dl: Effect of derivatisation of .thasone. Arch OpthalmoI1972, 88: 308-1l.