Investigative Ophthalmology & Visual Science, Vol. 32, No. 11, October 1991Copyright © Association for Research in Vision and Ophthalmology

Pharmacokinetic Basis for Nonadditivity of IntraocularPressure Lowering in Timolol Combinations

Vincent H. L. Lee, Amy M. Luo, Shaoyong Li, Samir K. Podder, Jomes Shih-Chieh Chang,Shigehiro Ohdo, and George M. Grass

The authors determined whether the ocular absorption of topically applied timolol in the pigmentedrabbit was affected significantly by coadministration with either pilocarpine or epinephrine in the samedrop to explain the nonadditivity in intraocular pressure lowering (IOP) seen clinically. They instilled25 fi\ of 0.65% timolol maleate solution (equivalent to 0.5% timolol), both in the presence and absenceof 2.6% pilocarpine nitrate or 1% epinephrine bitartrate, into pigmented rabbit eyes. The time course oftimolol concentration in the conjunctiva, anterior sclera, corneal epithelium, corneal stroma, aqueoushumor, iris-ciliary body, and lens was monitored for 360 min by using reversed-phase high-perfor-mance liquid chromatography. The area under the timolol concentration-time curve in all but one of theanterior segment tissues was reduced by 20-50% (mean, 40%) when timolol was coadministered withpilocarpine and by 20-70% (mean, 42%) when timolol was coadministered with epinephrine. Such aneffect was not a result of alterations in corneal permeability or aqueous humor turnover rate, nor was itrelated to the extent of systemic absorption caused by pilocarpine and epinephrine. Rather, the reduc-tion in ocular timolol absorption may have been caused by the accelerated washout of timolol by tearsstimulated by the coadministered drugs and, to a lesser extent, by the loss of timolol through binding tothe increased amount of tear proteins induced by the coadministered drugs. Thus, the nonadditivity inIOP lowering from timolol-pilocarpine and timolol-epinephrine combinations is probably caused bychanges in precorneal timolol clearance. Invest Ophthalmol Vis Sci 32:2948-2957,1991

Combination or multiple drug therapy often is pre-scribed during the advanced stages of glaucoma whensingle-drug therapy is incapable of maintaining intra-ocular pressure (IOP) adequately. Typically, suchcombinations are created to provide an additive, coop-erative, or synergistic lowering of the IOP by usingtwo drugs that together affect both inflow and outflowmechanisms. Thus, timolol, which reduces aqueoushumor inflow,1 was used in combination with drugsthat enhance outflow, such as pilocarpine,2"4 epineph-rine,56 dipivefrin,27 and others.3'8"10 A similar strategyrecently was reported for betaxolol11"13 and levobu-nolol.14

The long-term effectiveness of multiple drug ther-apy is drug dependent. Thus, the IOP-lowering effectof timolol is enhanced consistently by pilocarpine15"20

From the Department of Pharmaceutical Sciences, University ofSouthern California, School of Pharmacy, Los Angeles, California.

Supported in part by a grant (EY7389) from the National Insti-tutes of Health, Bethesda, Maryland, and by the Gavin S. HerbertProfessorship.

Submitted for publication: January 9, 1991; accepted June 4,1991.

Reprint requests: Vincent H. L. Lee, PhD, University of South-ern California, School of Pharmacy, Department of Pharmaceuti-cal Sciences, 1985 Zonal Avenue, PSC 704, Los Angeles, CA90033.

but not by epinephrine and dipivefrin, its pro-drug.615'21'22 Moreover, wherever there is an additiveeffect, it is often less than the arithmetic sum of theIOP-lowering effects of the individual drugs. 3"5>15>23"25

This phenomenon may be attributed to the nature ofthe disease state, to the order of and the time intervalbetween the instillation of drops, or to drug interac-tions at the absorption site.26 However, none of thesefactors has been studied carefully to our knowledge.

Most of the attention on drug interactions in oph-thalmology has been directed toward those that affectthe receptors involved in the regulation of aqueoushumor dynamics.6'24'27'28 There are, however, few re-ports of drug interactions mediated at the level of tearflow, protein binding, loss of drug to the systemic cir-culation, corneal drug penetration, or intraoculardrug distribution. Any of these factors may alter thedrug concentration at its target site and may be signifi-cant, particularly when the alteration affects the por-tion of the dose-response curve where small changesin drug concentration may cause large changes in thedrug response.

We wished to determine whether the ocular absorp-tion of timolol from a 0.65% timolol maleate solution(equivalent to a 0.5% timolol solution) from the eyesof the pigmented rabbit was affected significantly bythe coadministration with either 2.6% pilocarpine ni-

2948

No. 11 OCULAR DRUG INTERACTIONS / Lee er ol 2949

trate or 1% epinephrine bitartrate in the same drop,thus explaining the nonadditivity in IOP loweringseen clinically.15"22 We also were interested in themechanisms by which possible changes in ocular ti-molol absorption were mediated.

Materials and Methods

Materials

Timolol maleate, epinephrine bitartrate, pilocar-pine nitrate, histamine HC1, and rabbit serum albu-min were purchased from Sigma (St. Louis, MO).Propranolol HC1 and triethylamine HC1 were pur-chased from Aldrich (Milwaukee, WI). Male Dutch-belted pigmented rabbits, weighing 2.5-3.0 kg, werepurchased from Irish Farm (Los Angeles, CA). Theanimals were housed in standard laboratory rabbitcages and were fed a regular diet with no restrictionson the amount of food or water consumed. All experi-ments conformed to the ARVO Resolution on theUse of Animals in Research.

Preparation of Drug Solutions

Drug solutions for ocular and systemic absorptionexperiments were prepared in 10 mM Tris buffer, ad-justed to pH 7.4 with 10 N NaOH, and rendered iso-tonic by adding NaCl. Solutions for corneal penetra-tion experiments were prepared in glutathione bicar-bonate Ringer's (GBR) solution.29 In this instance,0.2% sodium bisulfite was added to the solutions con-taining epinephrine to protect the drug from beingoxidized during the experiment. All solutions wereprepared immediately before each experiment. Solu-tions containing rabbit serum albumin were allowedto equilibrate at 37°C for at least 30 min before use.

High-Performance Liquid Chromatographic Assay

Timolol was quantified using reversed-phase high-performance liquid chromatography on a BeckmanUltrasphere ODS column (column size, 25 cm X 4.6mm; particle size, 5 /tm) fitted with a OD-GU RP185-^m precolumn (1.5 cm) (West Coast Scientific,Hayward, CA), as previously described.30 The systemconsisted of a SCL-6A system controller, two LC-6Apumps, a SIL-6A autoinjector, a SPD-6A spectropho-tometric detector, and a CR-3A integrator (Shi-madzu, Baltimore, MD). The mobile phase consistedof varying proportions of 10% acetonitrile in metha-nol and 0.2% triethylamine HC1 in 5% acetonitrile atpH 3. The proportion of organic phase was kept at40% for 8 min, changed from 40% to 50% for the next7 min, kept at 50% for another 2 min, decreased from50% to 40% for the next 2 min, and kept at 40% for thefinal 4 min. The flow rate was 1 ml min"1. Timolol

was monitored at 294 nm. The retention time was 6.0±0.5 min for timolol and 15.0 ± 0.5 min for propran-olol, the internal standard. Under these chromato-graphic conditions, neither pilocarpine nor epineph-rine appeared in the chromatogram. The sensitivity ofthe assay was better than 5 nmol with respect to timo-lol in a 2-ml plasma sample. The intra- and interrunvariations were 5% and 7.5%, respectively.

At the time of assay, an aqueous humor sample(about 80 /nl) was mixed with 40 ix\ of 25 ^g ml"1

propranolol HC1 and 80 yul of 0.1 N HC1 in acetoni-trile. After centrifugation, 80-120 ix\ of the superna-tant was injected into the chromatograph. Excised tis-sues were soaked in 1 ml of 0.6% HC1O4 at 8°C for 12hr. Thereafter, the sample was mixed with 0.1 ml of10 /xg ml"1 of propranolol HC1 solution and 0.5 ml of1 M ammonium acetate buffer (pH 9), extracted with8 ml of diethyl ether by vortexing for 3 min, and thencentrifuged at 1500 g for 10 min. The upper organiclayer was transferred to a 15-ml screw-capped conicalcentrifuge tube containing 200 ^1 of 0.2 N HC1, vor-texed for 3 min, and centrifuged at 1500 g for 10 min.The organic phase was discarded, and 100-120 fi\ ofthe aqueous phase, containing timolol and proprano-lol, was injected into the chromatograph. The sameprocedure was used to extract timolol from 2 ml ofplasma. The extraction efficiency was better than 85%for both ocular tissue and plasma samples.

Ocular Absorption of Topically Applied Timolol

Unanesthetized rabbits were placed in polycarbon-ate restraining boxes with no restriction in head or eyemovement. We instilled 25 Atl of the test solution di-rectly onto the cornea of both eyes of each rabbit,collecting in the cul-de-sac. At 5, 15, 30, 60, 90, 150,180, 240, or 360 min postdosing, the rabbit was killedwith an overdose of sodium pentobarbital (Eutha-6;Western Medical Supply, Arcadia, CA) administeredthrough a marginal ear vein. After thoroughly rinsingthe corneal and conjunctival surfaces with 1.17% KC1solution and blotting them dry, the corneal epithe-lium was scraped with a no. 11 scalpel. Approxi-mately 150-200 /xl of aqueous humor was aspiratedfrom the anterior chamber using a 1-ml tuberculinsyringe fitted with a 27-gauge needle. The cornealstroma was excised by cutting at the corneolimbalmargin. The iris and ciliary body were difficult to sepa-rate from each other, and they were removed as onepiece. The lens was separated from the vitreous,which was discarded. The anterior sclera was dis-sected and trimmed to remove other tissue fragments.Finally, 8-10-mm tangential sections of conjunctivawere dissected from the upper and lower eyelids witha scalpel. Dissection of both eyes was completed

2950 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Ocrober 1991 Vol. 32

within 10 min. All excised tissues were rinsed withice-cold KG solution, blotted dry, transferred to pre-weighed microcentrifuge tubes containing 200 /xl of0.6% HC1O4, and stored at -70°C. At least four eyeswere used per time point.

Systemic Absorption of Topically Applied Timolol

The dosing procedure was as described in the ocu-lar absorption experiments. Fifteen minutes beforesolution instillation, the rabbits were cannulated in acentral ear artery using polyethylene tubing (PE-50;Intramedic) and heparinized with 1000 units of so-dium heparin (Western Medical Supply). Thereafter,25 /ul of a dosing solution was instilled into each eye ofa given rabbit. At 0, 3, 6, 10, 15, 30, 45, 60, 90, and120 min postdosing, 4-ml blood samples were col-lected into heparinized tubes. The volume of bloodaspirated was replenished with 50 ml of lactatedRinger's solution (Travenol, Deerfield, IL) throughan intravenous drip during the experiment. Prelimi-nary experiments revealed that the plasma timololconcentration fell below the detection limit after 120min; therefore, blood was collected until then. Theblood was centrifuged at 1500 g for 10 min to yield 2ml of plasma, quickly frozen, and stored at -20°Cuntil assayed. At least four rabbits were used.

Influence of Epinephrine on the Systemic Absorptionof Timolol From the Conjunctival and Nasal Mucosae

We wished to determine whether the effect of epi-nephrine on the systemic absorption of timolol wasmediated at the conjunctival or the nasal mucosa. Ab-sorption from the conjunctival mucosa was deter-mined by instilling 25 /M\ of a 0.65% timolol maleatesolution with and without 1 % epinephrine bitartratein rabbits whose conjunctival sac was isolated fromthe nasolacrimal duct by a punctum plug. This plugwas fashioned from a 5-mm segment of a polyethyl-ene tubing (PE 50; outer diameter, 1 mm) that hadbeen heat sealed at one end and beveled at the other.31

Absorption from the nasal mucosa was determined byinstilling 25 ix\ of a 0.65% timolol maleate solutionnasally through a polyethylene catheter (PE 50; outerdiameter, 1 mm) preinserted 15-20 mm into the lacri-mal sac and nasolacrimal duct of the same group ofrabbits after a 7-day washout period. In each case,serial blood samples were collected from each rabbituntil 120 min had elapsed and assayed for timolol asdescribed earlier. Immediately after the last sampling,the rabbits in the conjunctival-administration experi-ment were killed, and their anterior segment tissueswere excised and assayed for timolol as described ear-lier. At least four rabbits were used per condition.

Influence of Pilocarpine and Epinephrine onSystemic Timolol Pharmacokinetics

In this experiment, we tried to determine whetherchanges in the plasma timolol concentration-timeprofiles seen in timolol-pilocarpine combinationsand timolol-epinephrine combinations, comparedwith timolol instillation, were caused by changes inthe plasma clearance of timolol. We injected 50 ix\ of a0.65% timolol maleate solution with and without2.6% pilocarpine nitrate or 1% epinephrine bitartrateinto the marginal ear vein of pigmented rabbits. Thesampling schedule and sample processing procedurewere the same as in the topical solution-instillationexperiment described previously. At least four rabbitswere used per dosing time.

Influence of Histamine and Rabbit Serum Albuminon the Ocular and Systemic Absorption of TopicallyApplied Timolol

We wished to determine whether the ocular andsystemic absorption of topically applied timolol wasaffected by the coadministration of 0.5% histamineHC1 and 7% rabbit serum albumin. The dosing, sam-pling, and sample processing procedures were as de-scribed earlier for the ocular and systemic absorptionexperiments, except that ocular absorption was evalu-ated only at 30 min postdosing.

Influence of Pilocarpine and Epinephrine on theCorneal Penetration of Timolol

In this experiment, we tried to determine whetherchanges in the ocular absorption of topically appliedtimolol in the presence of either pilocarpine or epi-nephrine were caused by changes in corneal perme-ability. The rabbits were killed by a rapid injection ofsodium pentobarbital (Eutha-6) into the marginal earvein. The eyes were enucleated immediately with theconjunctiva and lids intact. The corneas were excisedand mounted in a Lucite-block perfusion chamber.29

We added 2.5 ml of GBR solution29 to the endothelialside, and an equal volume of the same solution con-taining drug was added immediately to the epithelialside. Mixing in each chamber was achieved by bub-bling a mixture of 95% O2 and 5% CO2. Thechambers' temperatures were kept at 37°C by a circu-lating water bath. At predetermined times up until240 min, 50-/ul aliquots were sampled from the endo-thelial side and immediately replaced by an equal vol-ume of GBR solution. The aliquots were assayed fortimolol by reversed-phase high-performance liquidchromatography. After correcting for the corneal sur-face area (1.075 cm2) and initial timolol concentra-

No. 11 OCULAR DRUG INTERACTIONS / Lee er ol 2951

tion in the donor reservoir, the apparent corneal per-meability coefficient was calculated from the slope ofthe linear portion of a plot of amount of timolol in thereceiver compartment versus time. At least four eyeswere used per condition.

Data Analysis

The data in the ocular and systemic absorption ex-periments were plotted as timolol concentration ver-sus time and fitted to a one-compartment pharmaco-kinetic model.32 The following pharmacokinetic pa-rameters were obtained: apparent absorption rateconstant (ka), apparent elimination rate constant (kg),peak timolol concentration (Cmax), peak time (tmax),volume of distribution, area under the concentration-time curve (AUC), and mean residence time (MRT).The plasma data from the intravenous administrationexperiment were found to obey the two-compartmentmodel better than the one-compartment model. Thefollowing two-compartment pharmacokinetic param-eters were obtained: k12, the rate constant for drugtransfer from the central to the peripheral compart-ment; k21, the rate constant for the opposite direction;k10, the rate constant for drug elimination from thecentral compartment; Vc, the volume of the centralcompartment, and Vdss, the volume of distribution atsteady state.

Results

Both pilocarpine and epinephrine reduced the Cmax

and AUC of timolol in all anterior segment tissues byapproximately 50% (Figs. 1A-G, Table 1). The excep-tion was the conjunctiva, in which the Cmax was two-fold higher and the AUC was sixfold higher when ti-molol was coadministered with epinephrine. The in-crease in timolol concentration in the conjunctiva didnot, however, lead to a corresponding increase in theunderlying sclera (Figs. 1A-B). Neither pilocarpinenor epinephrine affected the rank order of timololconcentration in the anterior segment tissues. Timo-lol concentration was the highest in the corneal epithe-lium, followed by the iris-ciliary body, cornealstroma, conjunctiva, aqueous humor, sclera, and lensin that order (Figs. 1A-G, Table 1).

Under all experimental conditions, timolol con-centration reached its peak in the conjunctiva, cor-neal epithelium, and corneal stroma relatively early (5min) compared with the aqueous humor (15 min),lens (90-150 min), and iris-ciliary body (240 min)(Figs. 1A-G, Table 1). Surprisingly, the peak time oftimolol concentration in the sclera (30-60 min) wasmuch longer than that in the overlying conjunctiva (5min) from which the sclera derived its drug. As can be

seen in Table 1, the long peak time in the sclera wasprobably a result of its slower rate of elimination (kg)in the sclera than that in the conjunctiva.

Pilocarpine did not affect the peak time of timololconcentration in any anterior segment tissues exceptthe sclera, which showed a decrease. Epinephrine pro-longed the peak time in the conjunctiva, corneal epi-thelium, and corneal stroma and shortened it in thesclera, iris-ciliary body, and lens. It did not affect it inthe aqueous humor (Table 1). Prolongation of thepeak time in the conjunctiva was probably a result ofthe vasoconstrictive effect of epinephrine.

The MRT of timolol in most anterior segment tis-sues was not altered by coadministration with eitherpilocarpine or epinephrine. This is consistent with thegeneral lack of change in the magnitude of ka and kg(Table 1), which are related to the MRT through therelationship: MRT = l/ka + 1/1 .̂

Although both pilocarpine and epinephrine re-duced the AUCs of timolol in the anterior segmenttissues, they differed on how they affected the rate andextent of timolol absorption in plasma (Fig. 1H). Spe-cifically, pilocarpine affected none of the pharmacoki-netic parameters of timolol in plasma except ka (Table1), whereas epinephrine reduced the Cmax and AUC oftimolol by 72% and 45%, respectively, and increasedits MRT 2.2 times (Table 1). The increase in MRT byepinephrine was caused by reduction in ka rather thanke; the MRT of timolol in the direct intravenous ad-ministration experiment was unaffected by coadmin-istration with epinephrine (Fig. 2, Table 2). Reduc-tion of systemic timolol absorption by epinephrinewas related to its vasoconstricting effect at both thenasal and conjunctival mucosa (Fig. 3, Table 3). Inthe presence of epinephrine, the systemic absorptionof timolol from the nasal and the conjunctival mu-cosa was 72% and 50% of control based on the AUCand 40% and 33% based on Cmax. The timolol concen-tration in the anterior segment tissues at 120 min afterconjunctival administration, however, was 1.6-7.1-fold higher in the presence of epinephrine than in itsabsence (Table 4).

Pilocarpine affected the plasma timolol AUC in theintravenous experiment differently than it did in thetopical administration experiment. The plasma timo-lol AUC was reduced by intravenously administeredpilocarpine (Table 2) but was unaffected by topicallyapplied pilocarpine (Table 1).

The net effect of coadministration of timolol withpilocarpine or epinephrine was a 50% reduction or nochange in the therapeutic index of timolol, respec-tively. Therapeutic index is defined as the ratio ofaqueous humor-plasma AUCs or iris-ciliary body-plasma AUCs. The AH-plasma ratio was 266 in the

25 n B

50 100Minutes

Fig. 1. Concentration-time profiles of timolol in the conjunctiva (plot A), anterior sclera (plot B), corneal epithelium (plot C), cornealstroma (plot D), aqueous humor (plot E), iris-ciliary body (plot F), lens (plot G), and plasma (plot H) of the pigmented rabbit following thetopical instillation of 25 nl of 0.65% timolol maleate solutions alone (O) or in combination with 2.6% pilocarpine nitrate (•) or 1% epinephrinebitartrate (•) . Error bars represent standard error of the mean for n = 4-8 for each experimental condition.

No. 11 OCULAR DRUG INTERACTIONS / Lee er ol 2953

Table 1. Influence of 2.6% pilocarpine nitrate and 1% epinephrine bitatrrate on the pharmacokineticparameters of timolol in plasma and various anterior segment tissues following topical instillationof 25 ul of 0.65% timolol maleate solutions to each eye

Parameter*

ka (min"1)

ke(102min"')

tmax (min)

(Mg/ml or Mg/g)

AUC(ng X min/ml orjig X min/g)

MRT (min)

Additive

None

Pilocarpine

Epinephrine

None

Pilocarpine

Epinephrine

None

Pilocarpine

Epinephrine

None

Pilocarpine

Epinephrine

None

Pilocarpine

Epinephrine

None

Pilocarpine

Epinephrine

Plasma

2.21(0.24)*0.82

(0.25)0.15

(0.015)2.28

(0.08)2.07

(0.10)0.63

(0.015)4.67

(1.44)13.00(0.62)44.00

(16.52)0.051

(0.006)0.050

(0.065)0.014

(0.002)1.59

(0.18)1.77

(0.35)0.87

(0.12)44.16(2.13)48.40(2.91)

142.04(25.38)

Co«/f

- §

0.21

0.18

- §

4.42

1.51

5

5

15

9.38(1.19)6.12

(0.75)20.66(2.86)

271.1

359.8

1699.9

86.4

68.2

78.9

Sclera

0.077

0.12

0.13

0.40

0.47

0.51

60

45

30

0.42(0.11)0.26

(0.06)0.34

(0.06)100.1

51.1

68.4

225.4

224.8

21.4.3

CE

0.50

0.72

0.95

1.34

1.42

1.14

5

5

15

157.13(7.75)92.29

(23.76)118.15(41.48)

14227

7400

11587

88.4

79.1

103.6

CS

0.62

3.40

0.31

2.21

1.61

1.16

5

5

15

21.33(3.75)18.03(1.43)13.80(5.06)

1892

1200

1317

124.8

74.8

92.6

AH

0.14

0.21

0.72

0.88

0.80

0.56

15

15

15

2.93(0.57)1.76

(0.23)1.35

(0.32)434.2

235.3

231.3

177.9

164.8

169.2

ICB

0.0062

0.0080

0.0061

0.36

0.42

0.45

240

240

150

69.74(11.3)37.82(3.02)25.38(2.89)

16686

10100

8319

206.7

197.3

199.0

Lens

0.0088

0.0296

0.0159

0.35

0.27

0.40

150

90

90

0.27(0.11)0.17

(0.09)0.085

(0.01)136.0

82.9

38.2

469.3

459.5

438.8

* See Methods for explanation of abbreviations.t Conj = conjunctiva, CE = corneal epithelium, CS = corneal stroma, AH

= aqueous humor, ICB = iris-ciliary body.

t Figures in parentheses represent standard error of the mean, n = 4.§ Cannot be estimated from data.

presence of epinephrine and 132 in the presence ofpilocarpine compared with 273 for the control. Theiris-ciliary body-plasma ratio was 9562 in the pres-ence of epinephrine and 5706 in the presence of pilo-carpine compared with 10,494 for the control.

As shown in Table 5, neither pilocarpine nor epi-nephrine affected the corneal penetration of timololin vitro. By contrast, the antioxidant sodium bisulfitereduced the corneal permeability coefficient of timo-lol 1.5-fold.

Table 2. Influence of 2.6% pilocarpine nitrate and 1% epinephrine bitartrate on the pharmacokineticparameters of timolol in plasma following intravenous bolus administration of 50 /il of 0.65% timololmaleate solutions

Parameter* Timolol alone With pilocarpine With epinephrine

kl2(102min-')K21 (102min-')kl0(102min-')Vc(L)Vdss (L)AUC (Mg X min/ml)MRT (min)

5.79 ± 2.56f§5.20 ± 1.34§5.30 ± 0.89*1.54±0.56§3.48 ± 1.79§3.40 ± 0.48*

31.9 ±2.6§

7.25 ± 0.95§4.33 ± 1.30§8.30 ± 1.76*2.29 ± 0.76§6.03 ± 0.63§1.66 ±0.32*

31.5 ±4.9§

7.58 ± 1.49§3.61 ±0.60§

11.12 ± 1.27*1.04 ± 0.30§3.32 ± 1.16§2.47 ± 0.35*

24.7 ±4.0§

* See Methods for explanation of abbreviations.t Mean ± SEM, n = 4.* Significantly different from timolol alone at P < 0.035 by one-way

ANOVA.§ Not significantly different at P = 0.2 by one-way ANOVA.

2954 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Ocrober 1991 Vol. 32

"Sb

30 60Minutes

90 120

Fig. 2. Effect of 2.6% pilocarpine nitrate and 1% epinephrinebitartrate on the concentration-time profiles of timolol in theplasma of the pigmented rabbit following the intravenous adminis-tration of 50 n\ of 0.65% timolol maleate solutions. Error bars repre-sent standard deviation for n = 4 for each experimental condition.• = timolol alone; O = timolol plus pilocarpine; A = timolol plusepinephrine.

Both 0.5% histamine and 7% rabbit serum albuminreduced timolol concentrations in the corneal stromaand iris-ciliary body at 30 min postdosing by approxi-mately 44% and 16%, respectively (Table 6). The re-duction in timolol concentrations in the conjunctiva,sclera, and lens was even more extensive (Table 6).Only 0.5% histamine reduced the timolol concentra-tions in the corneal epithelium and aqueous humor(Table 6). One-way analysis of variance revealed thatneither treatment affected the systemic bioavailabilityof topically applied timolol (P = 0.05). The AUC was1.67 ± 0.31 /xg • min/ml in the presence of 0.5% hista-mine and 1.53 ± 0.31 jug- min/ml in the presence of7% rabbit serum albumin, compared with 1.59 ± 0.35/xg • min/ml for the control.

120

Minutes

Fig. 3. Effect of 1% epinephrine bitartrate on the concentration-time profiles of timolol in the plasma of the pigmented rabbit follow-ing conjunctival and nasal administration of 25 ^1 of 0.65% timololmaleate solutions. Error bars represent standard error of the meanfor n = 4 for each experimental condition. D = conjunctival admin-istration without epinephrine; • = conjunctival administrationwith epinephrine; O = nasal administration without epinephrine; •= nasal administration with epinephrine.

Discussion

A significant finding in our study was that the coad-ministration of 0.65% timolol maleate with either2.6% pilocarpine nitrate or 1% epinephrine bitartrateresulted in a 50% reduction in its extent of absorptioninto the eye (Figs. 1A-G, Table 1). It thus follows thatunless the ocular absorption of pilocarpine or epineph-rine is increased by timolol, an unlikely situation, thenonadditivity in intraocular pressure lowering seen intimolol combinations with pilocarpine or epineph-rine515'23"25 is caused, at least in part, by a reduction intimolol absorption by the coadministered drug. Thereduction in systemic absorption of timolol by epi-

Table 3. Influence of 1% epinephrine bitartrate on plasma timolol pharmacokinetics following the instillation of0.65% timolol maleate solutions ocularly, conjunctivally, and nasally in the pigmented rabbit

Parameter*

Cma% fog/ml)

Uax (min)

ka(102min-')

ke(102min~')

AUC (Mg X min/ml)

MRT (min)

Epinephrine^

_

+-+-+-+-+—+

* See Methods for explanation of abbreviations.

Ocular

0.0510.0144.67

44.00221

15.02.280.631.590.87

44.16142.04

± 0.006t± 0.002± 1.44± 16.52± 2 4± 1.5± 0.08± 0.02± 0.18± 0.12± 2.13± 25.38

i Mean ± SEM,

Conjunctival

0.015 ±0.005 ±6.00 ±

48.75 ±57.7 ±

2.3 ±1.7 ±1.1 ±0.72 ±0.36 ±

40.23 ±67.75 ±

n = 4.

0.0010.00103.75

14.30.50.080.50.060.082.153.47

Nasal

0.0650.0266.33

24.0053.659.04.72.31.821.31

27.2146.33

± 0.008± 0.006± 2.03± 6.00±22.7± 0.5± 0.01± 0.5± 0.20± 0.10± 0.74± 2.75

f - = without epinephrine; -t- = with epinephrine.

No. 11 OCULAR DRUG INTERACTIONS / Lee er ol 2955

Table 4. Effect of 1% epinephrine bitartrate ontimolol concentrations in anterior segmenttissues at 120 min post-dosing followingconjunctival application

Timolol concentration (ng/g or ng/ml)*

Tissue/fluid Without epinephrine With epinephrine

ConjunctivaScleraCorneal epitheliumCorneal stromaAqueous humorIris-ciliary bodyLens

1.75 ± 0.190.44 ± 0.08

159.2 ± 15.816.1 ± 1.852.22 ± 0.21

77.61 ± 13.280.15 ± 0.03

12.46 ± 2.09f(7.1)$2.32 ± 0.28| (5.2)$

461.4 ±64.5|(2.9)$50.15 ± 7.58f(3.1)$4.14 ± 0.48t(1.9)$

126.35 ± 12.91| (1.6)$0.48 ± 0.09f(3.3)$

* Mean ± SEM, n = 8.t Statistically different from control at P < 0.02 by Student's t-test.$ Factor of increase over control.

nephrine (Fig. 1H) did not lead to an increase in theextent of ocular absorption (Figs. 1A-G). This findingwas similar to that of others,33 who pretreated the rab-bit eye with 50 /il of 2% epinephrine 5 min beforeinstilling timolol solution, and consistent with thelarger role played by the nasal than the conjunctivalmucosa in systemic timolol absorption.31 Only whenaccess of the administered dose to the nasal cavity wasdenied was there an increase (about twofold) in theocular tissue concentrations by the coadministeredepinephrine (Table 4).

The reduction in timolol concentrations in the ante-rior segment tissues caused by pilocarpine and epi-nephrine may be attributed to changes in precornealdrug loss, corneal drug penetration, or drug elimina-tion from the target tissue or fluid. The last possibilitycan be excluded because no changes are seen in the kgof timolol when coadministered with pilocarpine orepinephrine, even though both drugs decrease out-flow resistance and, hence, aqueous humor turnoverrate34 (Table 1). The second possibility—changes incorneal drug penetration—can also be excluded be-cause the in vitro corneal penetration experiments re-vealed no change in the corneal permeability coeffi-cient of timolol by pilocarpine or epinephrine (Table5). Therefore, the most probable explanation for thereduction in ocular timolol concentrations waschanges in precorneal clearance of timolol caused bypilocarpine and epinephrine, a major component ofwhich is solution drainage.35

The approximately equal extent of reduction in ti-molol concentrations in all anterior segment tissues inthe presence of either pilocarpine or epinephrine (Ta-ble 1) supports the notion of accelerated precornealclearance of timolol by the coadministered drugs. Al-though, for assay-sensitivity reasons, the precorneal

clearance of timolol was not measured, there is evi-dence that both pilocarpine and epinephrine can stim-ulate tear production,36"38 thus indirectly increasingthe solution drainage rate.39 Moreover, both choliner-gic and adrenergic stimulation are known to increasetear protein secretion,40"42 thereby potentially increas-ing loss of timolol through binding to the increasedamount of tear proteins. Others showed that timololat 0.1 Mg ml"1 is 60% bound to serum proteins.43 Nev-ertheless, under our experimental conditions, loss oftimolol to protein binding probably played only amodest role in reducing ocular timolol absorption. Asshown in Table 6, the extent of ocular timolol absorp-tion from a solution containing 7% rabbit serum albu-min was as high as 85% of the control on average. Arecent finding of the lack of statistically significantdifference in the aqueous humor timolol and pilocar-pine concentrations between the single drug prepara-tions and their fixed-ratio combination in anesthe-tized albino rabbits44 lends credence to this hypothesisof accelerated precorneal clearance of timolol by pilo-carpine- and epinephrine-induced lacrimation. Be-cause tearing is inhibited by anesthesia,45 pilocarpinewould not be able to express its lacrimation effect inthis previous study.44

In addition to increasing the tear flow and proteinsecretion rates, pilocarpine can reduce timolol con-centration in the conjunctival sac through its vasodi-lation effect. Such an effect increased the clearance ofglycerin from the conjunctival sac of anesthetizedrabbits by approximately threefold.35 The precornealclearance of timolol may be affected similarly in lightof the 40% reduction in the extent of ocular timololabsorption by 0.5% histamine, a potent vasodilator(Table 6). By contrast, the systemic absorption of ti-molol was not affected in either instance or when ti-molol was coadministered with pilocarpine. This isnot surprising because timolol is 80% absorbed sys-

Table 5. Influence of 2.6% pilocarpine nitrate,0.2% sodium bisulfite (SBS), and 1% epinephrinebitartrate on the corneal permeabilitycoefficient (Papp) of 0.65% timolol maleatein the pigmented rabbit

Additive Papp of timolol (106 cm/sec)*

None 12.27 ±0.84Pilocarpine 10.56 ± 0.67fSodium bisulfite 4.93 ± 0.31Epinephrine/sodium bisulfite 4.82 ± 0.33$

* Mean ± SD, n = 4.t Not statistically significant from timolol alone by Student's t-test (P

> 0.05).$ Not statistically significant from timolol plus sodium bisulfite by Stu-

dent's t-test (P > 0.05).

2956 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1991 Vol. 02

Table 6. Influence of 7% rabbit serum albumin and 0.5% histamine on timolol concentrations in ocular tissuesat 30 min following the topical instillation of 25 /A of 0.65% timolol maleate solutions in the pigmented rabbit

Tissue/fluid

ConjunctivaAnterior scleraCorneal epitheliumCorneal stromaAqueous humorIris-ciliary bodyLens

Control

3.66 ± 1.180.39 ± 0.08

116.37 ±25.012.64 ± 2.032.42 ± 0.35

16.27 ± 4.850.11 ± 0.05

Timolol concentration fog ml~' or ng g ')*

7<

1.360.04

115.219.302.22

12.07

Vo Albumin

± 0.33 (37%)tt± 0.04(9%)$± 16.22 (99%)± 1.91 (74%)$± 0.15(92%)± 1.96(74%)$0 (0%)$

0.5% Histamine

1.31 ±0.59(36%)$0.12 ±0.04(30%)$

57.83 ± 1.28(50%)$5.92 ±0.95(47%)$1.83 ±0.36(76%)$8.72 ±0.50(54%)$0.008 ± 0.008 (7%)$

* Mean ± SEM, n = 3-6.t Percent of control.

$ Significantly different from control at P < 0.05 by Student's t-test.

temically.31 Moreover, there is the possibility that ti-molol was diluted and washed out of the nasal cavitymore rapidly by the increased lacrimation rate.

Apparently, the vasoconstrictive effect of epineph-rine, which theoretically can increase the amount ofdrug in the conjunctival sac for ocular absorption, ismore than offset by its lacrimation effect. This is indi-cated by the 50% reduction in ocular absorption oftimolol when coadministered with epinephrine (Figs.1A-G, Table 1). Because of the rapid washout of ti-molol into the nasal cavity, reduction in the systemicabsorption of timolol by epinephrine must be a resultmore of its vasoconstrictive effect on the nasal mu-cosa than on the conjunctival mucosa, even thoughepinephrine was somewhat less effective in reducingsystemic timolol absorption when applied directly tothe nasal mucosa (Fig. 3, Table 3). A minor contribut-ing factor to the reduction in the plasma timolol AUCby epinephrine was an increase in the elimination rateconstant (k10) of timolol from plasma by epinephrine,as shown in the intravenous administration experi-ment (Table 2). In this instance, the plasma timololAUC in the presence of epinephrine was only 73% ofthe control.

In summary, the possibility that the ocular absorp-tion of one drug may be affected by a coadministereddrug must be considered in evaluating ophthalmicdrug candidates for use in combinations, particularlywhen the precorneal clearance, corneal permeability,and intraocular distribution of one drug are likely tobe affected by the other drug. This is the case whentimolol is coadministered with either pilocarpine orepinephrine. There is, therefore, a pharmacokineticbasis for the nonadditivity in intraocular pressure low-ering seen clinically when these drugs are used in com-bination to treat advanced glaucoma.

Key words: ocular drug interactions, ocular drug pharmaco-kinetics, precorneal drug clearance, timolol-epinephrinecombinations, timolol-pilocarpine combinations

References

1. Neufeld AH, Bartels SP, and Liu JHK: Laboratory and clinicalstudies on the mechanism of action of timolol. Surv Ophthal-mol 28:286, 1983.

2. O'Connor MA and Mooney DJ: The additional pressure-lowering effect in patients with glaucoma of pilocarpine 2%,adrenaline 1%, or guanethidine 3% with adrenaline 0.5% andtimolol 0.25%: A double-blind cross over study. Trans Ophthal-molSocUK 103:588, 1983.

3. Lofors KT, Hovding G, Viksmoen L, Assved H, Bergaust B,and Bulie T: Twelve-hour IOP control obtained by a singledose of timolol/pilocarpine combination eye drops. Acta Oph-thalmol (Copenh) 68:323, 1990.

4. Thygesen J: Comparison of timolol and pilocarpine combina-tion versus pilocarpine alone in the management of glaucoma:A randomized double-masked study. Int J Ophthalmol 7:57,1990.

5. Higgins RG and Brubaker RF: Acute effect of epinephrine onaqueous humor formation in the timolol-treated normal eye asmeasured by fluorophotometry. Invest Ophthalmol Vis Sci19:420, 1980.

6. Thomas JV and Epstein DL: Timolol and epinephrine in pri-mary open angle glaucoma: Transient additive effect. ArchOphthalmol 99:91, 1981.

7. Keates EU and Stone RA: Safety and effectiveness of concomi-tant administration of dipivefrin and timolol maleate. Am JOphthalmol 91:243, 1981.

8. van der Pol BAE and Wattel E: Timolol and aceclidine: A clini-cal study of interaction in the eye. Acta Ophthalmol (Copenh)63:411, 1985.

9. Mills KB, Vogel R, and Jacobs N: Timolol and guanethidineplus adrenaline in combination in the treatment of chronicopen angle glaucoma. Trans Ophthalmol Soc UK 104:621,1985.

10. Cerulli L, Corsi A, Pocobelli A, and Zapelloni A: Evaluation ofthe pharmacodynamic effects of dapiprazole and timolol aloneand in combination in healthy volunteers. Glaucoma 10:11,1988.

11. Berry DP, Van Buskirk EM, and Shields MB: Betaxolol andtimolol: A comparison of efficacy and side effects. Arch Oph-thalmol 102:42, 1984.

12. Allen RC and Epstein DL: Additive effect of betaxolol andepinephrine in primary open angle glaucoma. Arch Ophthal-mol 104:1178, 1986.

13. Weinreb RN, Ritch R, and Kushner FH: Effect of adding be-taxolol to dipivefrin therapy. Am J Ophthalmol 101:196,1986.

No. 11 OCULAR DRUG INTERACTIONS / Lee er ol 2957

14. David R, Ober M, Masi R, Elman J, Novack GD, Sears ML,and Batoosingh AL: Treatment of elevated intraocular pres-sure with concurrent levobunolol and pilocarpine. Can J Oph-thalmol 22:208, 1987.

15. Boger WP III, Puliafito CA, Steinert RF, and Langston DP:Long-term experience with timolol ophthalmic solution in pa-tients with open-angle glaucoma. Trans Am Acad OphthalmolOtolaryngol 85:259, 1978.

16. Kerty E and Horven I: Glaucoma treatment with timolol. ActaOphthalmol (Copenh) 56:705, 1978.

17. Obstbaum SA, Galin MA, and Katz IM: Timolol: Effect onintraocular pressure in chronic open-angle glaucoma. AnnOphthalmol 10:1347, 1978.

18. Nielsen NV and Eriksen JS: Timolol in maintenance treatmentof ocular hypertension and glaucoma. Acta Ophthalmol (Co-penh) 57:1070, 1979.

19. Ober M and Scharrer A: Timolol und parasympathicomime-tica bei de behandlung des erhohten intraocularen druckes.Graefes Arch Clin Exp Ophthalmol 211:59, 1979.

20. Zimmerman TJ and Canale P: Timolol: Further observations.Ophthalmology 86:166, 1979.

21. Goldberg I, Ashburn FS, Palmberg PF, Kass MA, and BeckerB: Timolol and epinephrine: A clinical study of ocular interac-tions. Arch Ophthalmol 98:484, 1980.

22. Cyrlin MN, Thomas JV, and Epstein DL: Additive effect ofepinephrine to timolol therapy in primary open angle glau-coma. Arch Ophthalmol 100:414, 1982.

23. Korey MS, Hodapp E, Kass MA, Glodberg I, Gordon M, andBecker B: Timolol and epinephrine: Long-term evaluation ofconcurrent administration. Arch Ophthalmol 100:742, 1982.

24. Schenker HI, Yablonski ME, Podos SM, and Linder L: Fluoro-photometric study of epinephrine and timolol in human sub-jects. Arch Ophthalmol 99:1212, 1981.

25. Keates EU: Evaluation of timolol maleate combination ther-apy in chronic open-angle glaucoma. Am J Ophthalmol88:565, 1979.

26. Chrai SS, Makoid MC, Eriksen SP, and Robinson JR: Dropsize and initial dosing frequency problems of topically appliedophthalmic drugs. J Pharm Sci 63:333, 1974.

27. Boas RS, Messenger MJ, Mittag TW, and Podos SM: The ef-fects of topically applied epinephrine and timolol on intraocu-lar pressure and aqueous humor cyclic-AMP in the rabbit. ExpEye Res 32:681, 1981.

28. Crawford K and Kaufman PL: Pilocarpine antagonizes prosta-glandin F2a. Arch Ophthalmol 105:1112, 1987.

29. O'Brien WJ and Edelhauser HF: The corneal penetration oftrifluorothymidine, adenine arabinoside and idoxuridine: Acomparative study. Invest Ophthalmol Vis Sci 16:1093, 1977.

30. Chang SC, Bundgaard H, Buur A, and Lee VHL: Low dose

O-butyryl timolol improves the therapeutic index of timolol inthe pigmented rabbit. Invest Ophthalmol Vis Sci 29:626,1988.

31. Chang SC and Lee VHL: Nasal and conjunctival contributionsto the systemic absorption of topical timolol in the pigmentedrabbit: Implications in the design of strategies to maximize theratio of ocular to systemic absorption. J Ocul Pharmacol 3:159,1987.

32. Gibaldi M and Perrier D: Pharmacokinetics, 2nd ed. NewYork, Marcel Dekker, 1982, pp. 1-42.

33. Kyyronen K and Urtti A: Effects of epinephrine pretreatmentand solution pH on ocular and systemic absorption of ocularlyapplied timolol in rabbits. J Pharm Sci 79:688, 1990.

34. Kaufman PL and Barany EH: Loss of acute pilocarpine effecton outflow facility following surgical disinsertion and retrodis-placement of the ciliary muscle from the scleral spur in thecynomolgus monkeys. Invest Ophthalmol 15:793, 1976.

35. Lee VHL and Robinson JR: Mechanism and quantitative eval-uation of pilocarpine disposition in the albino rabbit eye. JPharm Sci 68:673, 1979.

36. de Haas EBH: Lacrimal gland response to parasympathomi-metics after parasympathetic denervation. Arch Ophthalmol64:34, 1960.

37. Boissier JR, Advenier CH, and Ho S: Secretion lacrymale chezle lapin et recepteurs j8-adrenergiques. J Pharmacie 7:241,1976.

38. Aberg G, Adler G, and Wikberg J: Inhibition and facilitation oflacrimal flow by /J-adrenergic drugs. Acta Ophthalmol (Co-penh) 57:225, 1978.

39. Conrad J, Reay WA, Polcyn RE, and Robinson JR: Influenceoftonicity and pH on lacrimation and ocular drug bioavailabil-ity. J Parenter Sci Assoc 32:149, 1978.

40. Bromberg BB: Autonomic control of lacrimal protein secre-tion. Invest Ophthalmol Vis Sci 20:110, 1981.

41. Friedman Z, Lowe M, and Selinger Z: Beta adrenergic recep-tors stimulated peroxidase secretion from rat lacrimal gland.Biochim Biophys Acta 675:40, 1981.

42. Dartt DA, Ronco LV, Murphy SA, and Unser MF: Effect ofphorbol esters on rat lacrimal gland protein secretion. InvestOphthalmol Vis Sci 29:1726, 1988.

43. Belpaire FM, Bogaert MC, and Rosseneu M: Binding of |8-adrenoceptor blocking drugs to human serum albumin, to ar

acid glycoprotein and to human serum. Eur J Clin Pharmacol22:253, 1982.

44. Ellis PP, Wu PY, and Riegel M: Aqueous humor pilocarpineand timolol levels after instillation of the single drug or in com-bination. Invest Ophthalmol Vis Sci 32:520, 1991.

45. Chrai SS, Patton TF, Mehta A, and Robinson JR: Lacrimaland instilled fluid dynamics in rabbit eyes. J Pharm Sci62:1112, 1973.