Different effects of pH on the permeation of pilocarpine and pilocarpine prodrugs across the isolated rabbit cornea

European Journal of Pharmaceutical Sciences, 6 (1998) 169–176

Different effects of pH on the permeation of pilocarpine and pilocarpine prodrugs across the isolated rabbit cornea b ¨ Pekka Suhonen a , *, Tomi Jarvinen , Satu Koivisto a , Arto Urtti a a

b

Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland Received 26 August 1996; accepted 28 November 1996

Abstract Ocular absorption of pilocarpine and many other ophthalmic drugs can be improved by prodrug derivatization. For stability and solubility reasons basic prodrugs must be formulated at acidic pH, which may affect the corneal drug permeability. We studied the effects of pH on in vitro permeation of pilocarpine, pilocarpic acid benzyl diester prodrugs [O-propionyl (I) and O-valeryl (II)] and O,O9-(1,4-xylylene) bispilocarpic acid diester prodrugs [O,O9-diacetyl (III), O,O9-dipropionyl (IV) and O,O9-divaleryl (V)] through albino rabbit cornea. Reversed-phase high-performance liquid chromatography was used to assay pilocarpine and its prodrugs. The permeability coefficient for pilocarpine decreased more than three times, from 2.8310 26 cm / s to 0.9310 26 cm / s, when the pH was decreased from 7.65 to 5.5. At pH 7.65 permeability of pilocarpine improved several fold with delivery as prodrugs. Acidic pH (5.5, 6.0) affected to a different extent the corneal permeability of pilocarpine given as prodrugs. Consequently, the rank order of the corneal permeabilities among the compounds was different at various pH values. The effect of pH was greatest (an order of magnitude) for prodrugs with intermediate lipophilicity (I, III, IV), while pH had only minor or no effect on permeability of the most lipophilic prodrugs (II, V). In conclusion, the effect of pH on pilocarpine delivery as prodrug is dependent on prodrug structure and the advantage gained with prodrugs relative to pilocarpine is dependent on formulation pH.  1998 Elsevier Science B.V. Keywords: Pilocarpine; Prodrug; Pilocarpic acid diesters; Bispilocarpic acid diesters; pH; Corneal permeability; Drug delivery; Ocular absorption

1. Introduction The ocular activity, stability, and solubility of several basic drugs is affected by the pH of eyedrops (Anderson and Cowle, 1968; Gibbs and Tuckerman, 1974). For example, pilocarpine, a weak base, is therapeutically more active in neutral eyedrops than in acidic solutions. Higher pH values favor the unionized form of the drug (Anderson and Cowle, 1968), which penetrates the cornea more rapidly than ionized form (Sieg and Robinson, 1977). For stability reasons pilocarpine eye drops are adjusted to an acidic pH, which reduces the ocular bioavailability. Optimal drug absorption depends on achieving a rapid penetration rate across the cornea and the impact of pH on permeability (Francoeur et al., 1983, 1985). The physicochemical properties of the ionizable drug such as lipophilicity, molecular size, pKa value, and ionization state are the main factors affecting corneal permeability *Corresponding author. Tel.: 11 358 71162488; fax: 11 358 71162456; e-mail: [email protected] 0928-0987 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 97 )10002-1

(Schoenwald and Huang, 1983). Poor corneal permeability and low ocular bioavailability of topically applied pilocarpine are partly due to the low lipophilicity of the drug. The corneal permeability of pilocarpine can be increased by prodrugs that will release the active drug via hydrolysis in the cornea (Suhonen et al., 1991a). In the case of pilocarpine, lipophilic prodrugs, pilocarpic acid diesters (Scheme 1) and O,O9-(1,4-xylylene) bispilocarpic acid diester fumarates (Scheme 2), penetrate the cornea more easily than the parent compound and thereafter, pilocarpine is formed through a sequence of enzymatic and chemical hydrolysis (Bundgaard et al., 1986b; Suhonen et al., 1991b). Corneal permeabilities of prodrugs have been compared with parent drugs only at neutral pH values. Typically the stability of prodrugs in solutions is pHdependent: e.g. pilocarpine and timolol prodrugs, which have improved stability at acidic pH compared to neutral conditions. Permeability of an ionized drug in a membrane is determined by the permeabilities of ionized (PBH 1 ) and unionized (PB ) drugs, and by the fraction of each form in the solution. The latter is dependent on pH and pKa values.

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various lipophilicities, pilocarpic acid benzyl diesters and O,O9-(1,4-xylylene) bispilocarpic acid diesters through albino rabbit cornea.

2. Experimental procedures

2.1. Materials

Scheme 1. Structures of pilocarpic acid diester prodrugs.

The difference between PB and PBH 1 is very dependent on membrane and drug structure. Therefore, pH dependence in permeabilities of different compounds, e.g. in the cornea, might be different. For pilocarpine Mitra and Mikkelson (1988) determined PBH 1 and PB values in the 26 26 cornea as 4.81310 cm / s and 9.74310 cm / s, respectively. We studied the effects of pH on the in vitro permeabilities of pilocarpine, and of its prodrugs of

Pilocarpine hydrochloride was a gift from Leiras (Finland). Isopilocarpine nitrate was purchased from Aldrich (Germany). Pilocarpic acid diester fumarates (O-propionyl (I), Mr : 372, and O-valeryl (II), Mr : 400) and bispilocarpic acid diester fumarates (O,O9-diacetyl (III), Mr : 639, O,O9dipropionyl (IV), Mr : 667, and O,O9-divaleryl (V), Mr : 723) were synthesized and identified as described else¨ where (Jarvinen et al., 1991a,b), respectively. Molecular weights are expressed as free base. Structures of prodrugs are shown in Schemes 1 and 2. HPLC grade methanol was from Rathburn (Walkerburn, Scotland). All other chemicals were of analytical grade. Male and female albino rabbits (New Zealand White strain, 2.0–3.2 kg), were used. The animals were housed in standard laboratory rabbit cages and they were fed with regular diet with no restrictions on the amount of food or water consumed. All the experiments conformed to the ARVO resolution on the use of animals in research.

2.2. Apparent partition coefficients The lipophilicity of pilocarpine, pilocarpic acid diester fumarates and bispilocarpic acid diester fumarates was determined by monitoring the distribution of the compounds between 1-octanol and phosphate buffer using the ‘shake-flask’ method or by calculating the capacity factors of the compounds with reversed-phase high-performance ¨ liquid chromatography (RP-HPLC) (Jarvinen et al., 1991c,d). The apparent distribution coefficients at 258C were determined by dissolving the drug in the 0.16 M phosphate buffer phase (pH 4.2, 5.0, 6.0 and 7.4, and ionic strength of 0.5 adjusted with sodium chloride) and shaking continuously with 1-octanol at 258C for 60 min to reach a distribution equilibrium. The volumes of each phase were chosen so that the drug concentration in the aqueous phase, before and after distribution, could be measured by the RP-HPLC procedure. The apparent distribution coefficients were calculated according to the method described previ¨ ously (Jarvinen et al., 1991c,d). Triplicate determinations were carried out for each compound. The distribution coefficients (DC 7.65 ) for pilocarpine and prodrugs at pH 7.65 were calculated from the distribution coefficients at pH 7.4 according to Eq. (1): Scheme 2. Structures of O,O9-(1,4-xylylene)bispilocarpic acid diester prodrugs.

S

1 DC 7.65 5 DC 1 1 ]]]]]] antilog (pH 2 pKa )

D

(1)

P. Suhonen et al. / European Journal of Pharmaceutical Sciences 6 (1998) 169 – 176

2.3. Dissociation constants The dissociation constants (pKa values) of pilocarpine and pilocarpine prodrugs were measured by titration of 2 mM drug solutions in water–ethanol (50:50, v / v) with 0.5 M hydrochloric acid at room temperature (258C). Since the in vitro permeability experiments were performed at 378C, and the ionic strength of pilocarpine and prodrug solutions varied from 0.3 to 0.4, respectively, the pKa values ¨ obtained previously (Jarvinen et al., 1991d) were corrected to correspond the conditions in the permeability experiments. Due to the inadequate water-solubility of the prodrugs, the pKa values were determined in the presence of 50% ethanol. The pKa value of pilocarpine decreased from 7.0 to 6.3 in 50% ethanol. pKa values of the prodrugs in 50% ethanol were 5.8–6.2. We roughly estimate that in ¨ water the pK 9a values are 6.5–6.9 (Jarvinen et al., 1991d). ¨ According to the Debye–Huckel equation ionic strength affects the pK 9a values only 0.1 units. Consequently, the pK 9a values for pilocarpine and prodrugs decreased to a range of 6.4–6.9 at 378C and an ionic strength of 0.4 (Table 1).

2.4. Permeability experiments In order to monitor the condition of the cornea samples, mannitol, D-[1- 3 H(N)]-(sp act. 30 Ci / mmol; Du Pont, Boston, MA, USA), and progesterone, [4- 14 C]-(sp act. 57.2 mCi / mmol; Du Pont) were used as indicators of the corneal integrity. Samples of 200 ml volume were taken from the receiver chamber at fixed time intervals. Part of each sample (150 ml) was used for HPLC analysis and the rest (50 ml) was transferred to scintillation counting vials. Then, 450 ml of distilled water and 4.5 ml of scintillation cocktail (ACS, Amersham, Arlington Heights, IL, USA) were added. The samples were kept in the dark for 20 h in

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order to minimize chemiluminescence prior to counting in a scintillation counter. Count rates were obtained using a LKB Rack-beta 1216 liquid scintillation counter (Wallac, Turku, Finland). A counting time of 8 min and a counting limit of 90 000 were used. The prodrug solutions for corneal penetration experiments were prepared in glutathione bicarbonated Ringer’s (GBR) solution as described earlier (Suhonen et al., 1991b). The concentration of sodium bicarbonate was 0.570 g / l at reduced pH (,7.65) and 4.908 g / l at pH 7.65. The pH of the buffer solution was adjusted with a dilute HCl solution to acidic pH values. Solutions were stored in a refrigerator and they were used within 2 weeks. The rabbits were sacrificed and the cornea prepared as described before (Suhonen et al., 1991a). GBR solution was added first to the endothelial side (3.4 ml) to prevent the cornea from buckling, and immediately thereafter, 3.2 ml of the same solution containing 0.15–24 mM of pilocarpine, 0.06–1 mM of pilocarpic acid diester or 0.02– 12 mM of O,O9-(1,4-xylylene) bispilocarpic acid diester in GBR buffer, was added to the epithelial side. The concentrations of the drug in the donor side were chosen according to the solubility of the derivatives in the buffer. The pH of the buffer solutions was similar in both chambers. Mixing was achieved by bubbling a mixture of 95% O 2 and 5% CO 2 and the chambers were thermostated at 378C. Samples of 200 ml were withdrawn from the receiver side for 240 min and always immediately replaced by an equal volume of blank buffer. At the end of the experiment, the remaining prodrug and formed pilocarpine in the epithelial side were measured. Total absorption of prodrug into the cornea was determined as the percentage decrease of prodrug in the epithelial side. Pilocarpine, isopilocarpine, pilocarpic acid, and isopilocarpic acid were quantified using RP-HPLC on a deactivated LC18-DB Supelcosil column (Supelco, Belle-

Table 1 Logarithm of the apparent distribution coefficient (log DC) between 1-octanol and phosphate buffer at various pH values, pK a9 values, and f50% for pilocarpinc, pilocarpic acid benzyl diesters and O,O9-(1,4-xylylene) bispilocarpic acid diesters Compound

pK 9a e

Log (DC)

f50% (min)c (80% human serum a or plasma b

pH 4.2

pH 5.0

pH 6.0

pH 7.4

pH 7.65 d

21.74

21.73

21.28

0.01 a

0.08

6.9 a

Pilocaryic acid diesters I 0.76 II 1.89

1.40 2.52

2.24 2.99

3.30 a 4.43 a

3.36 4.48

6.8 a 6.7 a

26 a 31 a

O,O9 -(1.4 -xylylene) bispilocarpic acid diesters III 21.22 20.29 IV 20.44 0.66 V 1.64 2.96

1.37 2.64 4.66

3.04 b 4.08 b 6.87 b

3.08 4.10 6.91

6.6 b 6.4 b 6.6 b

32 b 24 b 42 b

Pilocarpine

a

¨ Data from Jarvinen et al., 1991a. ¨ Data from Jarvinen et al., 1991d. c f50% 5time, when 50% of total pilocarpine is formed. d Converted through Eq. (1) using experimental value at pH 7.4. e ¨ ¨ Data from Jarvinen et al., 1991a,d were converted to pK 9a values according to Debye–Huckel equation. b

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fonte, USA) (25034.6 mm) with a particle size of 5 mm at 408C. The mobile phase consisted of 5% KH 2 PO 4 (pH 2.5)–methanol (97:3, v / v) with a flow-rate of 1.5 ml / min. A deactivated Supelcosil LC8-DB column (Supelco) (15034.6 mm) with 5 mm particles was used for the determination of pilocarpine prodrugs. The solvent system was 0.02 M KH 2 PO 4 (pH 4.5)–methanol (29:71, v / v) with a flow-rate of 1.0 ml / min. HPLC analysis was carried out as described previously (Suhonen et al., 1991b). pH was measured with Orion SA 520 pH meter (Boston, USA) at the temperature of study. The condition of the isolated cornea was evaluated visually after the experiment. Some stromal swelling had occurred, as indicated by the partial clouding of the cornea. However, the linearity of the steady-state fluxes for each compound examined suggest that the rate-limiting corneal layers remained intact. After taking the corneal surface area (1.17 cm 2 ) and the initial drug concentration in the donor compartment into account, the apparent permeability coefficient (Papp ) was calculated from the slope of the linear portion of a graph showing the amount of pilocarpine, progesterone or mannitol in the receiver compartment vs. time (Suhonen et al., 1991a). The apparent corneal permeability (Papp ) at a given pH as a function of the permeabilities of ionized (Pi ) and unionized (Pu ) species is as: Papp 5 Pu fu 1 Pi fi

(2)

where Pu and Pi are the permeabilities of the unionized and ionized species, respectively, and fu and fi are the corresponding fractions. When fi 512fu , Eq. (2) can be rewritten as: Papp 5 Pi 1 fu (Pu 2 Pi )

(3)

When total permeability is plotted as a function of fraction of the unionized form, the y-intercept ( fu 50) is the permeability of the ionized form. The slope is the difference between the permeabilities of ionized and unionized forms (Pu 2Pi ) and at fu 51, the permeability is that of the unionized form (Pi ). For the prodrugs, the effect of pH on permeability can be expressed as: Papp 5 Pi Xi 1 fu (Pu Xu 2 Pi Xi )

and [ 14 C]-progesterone penetrates via transcellular route. A pH change from 7.65 to 5.0 did not affect the permeabilities of the markers. The permeabilities of [ 3 H]mannitol at pH 7.65 and 5.0 were (3.860.7)310 27 (n55) and (4.060.3)310 27 cm / s (n54), respectively. The permeability of [ 14 C]-progesterone was (9.561.6)310 27 cm / s (n54) at pH 7.65 and (8.960.4)310 27 cm / s (n54) at pH 5.0. Thus, changes in the pH in the range 5.0–7.65 do not affect the tightness of intercellular or transcellular route of the rabbit cornea. Consequently, the effects of pH on corneal permeability are not due to effects on corneal integrity. Fig. 1 shows the penetration of pilocarpine at various pH values across an excised cornea. Pilocarpine permeability in the cornea increases with increasing pH and fraction of the un-ionized form of pilocarpine as prodrug in the donor phase. As expected, the log (DC) of pilocarpine decreased with lower pH (Table 1). Following the lag time, a linear relationship between corneal permeation and time was observed. The corneal permeabilities of pilocarpine calculated according to Eq. (3) were 0.6310 26 cm / s for the ionized species (Pi ), and 3.2310 26 cm / s for the unionized species (Pu ). The ratio of Pu /Pi was about 5 (Fig. 2, Table 2). The corneal permeation of many pilocarpine prodrugs followed a similar trend to that of pilocarpine. The calculated corneal permeability of pilocarpine given as I increased from 2.1310 26 cm / s for BH 1 to 10.7310 26 cm / s for B. The ratio of un-ionized species to ionized species is the same as that of pilocarpine (|5) (Fig. 2, Table 2). Compound I was completely hydrolyzed in the cornea at pH 7.65, but at lower pH (,6.85) small fraction (,1%) of the prodrug penetrated intact through the cornea. During 4 h the fraction of prodrug absorbed to the cornea from the donor phase was 81% at pH 7.65 and 38% at pH 5.0. From pH 7.65 to 5.0 the value of log (DC) decreased from 3.4. to 1.4 (Table 1). Log (DC) of II decreased by 2.0 units, from 4.43 to the range 2.5–3.0, when pH was decreased from 7.4 to 5.5

(4)

where Xu and Xi are the fractions of unionized and ionized prodrug converted to pilocarpine in the cornea, respectively.

3. Results The effect of an acidic pH on the corneal barrier was evaluated in vitro by monitoring the permeability of hydrophilic [ 3 H]-mannitol and lipophilic [ 14 C]-progesterone. [ 3 H]-mannitol is a marker for intercellular penetration

Fig. 1. Permeability of pilocarpine at various pH values across an excised rabbit cornea. All points represent an average (6S.E.M.) of two to six corneas. The vertical bars that are absent were smaller than the symbol.

P. Suhonen et al. / European Journal of Pharmaceutical Sciences 6 (1998) 169 – 176

Fig. 2. Corneal permeability of pilocarpine (s), O-propionyl pilocarpic acid benzyl diester (I) (h), O-valeryl pilocarpic acid benzyl diester (II) (앳), O,O9-diacetyl (1,4-xylylene) bispilocarpic acid diester (III) (X), O,O9dipropionyl (1,4-xylylene) bispilocarpic acid diester (IV) (d), and O,O9divaleryl (1,4-xylylene) bispilocarpic acid diester (V) (m) as a function of the fraction un-ionized ( fu ) present. All points represent an average (6S.E.M.) of two to six corneas. When no error bar is indicated, the standard error of the mean is smaller than the size of the symbol used.

(Table 1), but its corneal permeability did not significantly change (Fig. 3). Lowering the pH from 7.65 to 5.50 decreased the fraction of prodrug absorption into the cornea from its epithelial side from 92% to 39%. The corneal permeability did not change remarkably with changing degree of ionization (Table 2). The apparent permeability coefficient of pilocarpine administered as III decreased 11 times from 6.5310 26 cm / s to 0.6310 26 cm / s, when pH was decreased from 7.65 to 5.50 (Fig. 3). Only ,1.2% of intact prodrug reached the endothelial side buffer solution after 4 h perfusion. At the same time, the fraction of prodrug absorption to the cornea was 42% at pH 7.65 and 5% at pH 5.50 (Fig. 5). The calculated permeability of ionized species was zero and that of un-ionized species was 7.13 10 26 cm / s. There were remarkable differences in pilocarpine permeability depending on the pH value. Pilocarpine permeability for compound IV decreased over 40 times at pH Table 2 Apparent corneal permeability coefficient of pilocarpine and its prodrugs, when the fraction of the compound is in entirely ionized (Pi ) or unionized (Pu ) form Compound Pilocarpine

Pu 310 26 (cm / s) 3.2

Pi 310 26 (cm / s)

Pu /Pi

0.6

5.3

2.1 7.3

5.1 1.1

O,O9 -(1,4 -xylylene) bispilocarpic acid diesters III 7.1 |0 IV 22.1 |0 V 13.1 8.3

2 2 1.6

Pilocarpic acid diesters I 10.7 II 8.1

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Fig. 3. Corneal permeability of pilocarpine and the prodrugs in vitro as a function of the pH of the buffer solution. Descriptions for the symbols as in Fig. 2. All points represent an average (6S.E.M.) of two to six corneas. When no error bar is indicated, the standard error of the mean is smaller than the size of the symbol used.

5.0 (0.5310 26 cm / s) compared to pH 7.65 (20.2310 26 cm / s) (Fig. 3). As pH decreased from 7.65 to 6.85, the corneal permeability coefficient was decreased only slightly, but at the more acidic range (pH 6) permeability decreased drastically (Fig. 3). Log (DC) of compound IV decreased by 3.4 units from 4.10 to 0.66 with decreasing pH (from 7.65 to 5.0) (Table 1). Lowering the pH from 7.65 to 5.00 decreased the prodrug absorption into the cornea during 4 h from 72% to 4%. For compound IV Pu and Pi were 22.0310 26 cm / s and zero, respectively (Fig. 2, Table 2). Pilocarpine penetration for V, the most lipophilic derivative studied, did not vary significantly at pH range of 7.65 to 6.00 (Fig. 3). Log (DC) decreased from 6.91 to 4.66 when pH was reduced from 7.65 to 6.0 (Table 1). At pH 6.85 and 6.00, 4% and 3% respectively, of intact prodrug reached the endothelial side. Pi and Pu differed only slightly from each other (Table 2). A linear correlation was found between log (DC) and the fraction of un-ionized form at each derivative, but at the same un-ionized values among pilocarpine and prodrugs, log (DC) differ even orders of magnitude (Fig. 4). A sigmoidal correlation was found between log (DC) and the corneal partitioning for pilocarpine prodrugs determined as the fraction of intact prodrug in the epithelial side at 4 h (Fig. 5). The corneal partitioning of pilocarpine and its prodrugs increased with decreasing ionization and increasing lipophilicity. The greatest relative changes in corneal absorption are seen at log (DC) values below 3.0–3.5 range. At high log (DC) values only slight changes in absorption are seen.

4. Discussion The cornea consists of three primary layers: the cellular epithelium and endothelium are lipophilic, while gel-like

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Fig. 4. Log apparent distribution coefficient of pilocarpine and the prodrugs as a function of the un-ionized fraction ( fu ) present. Descriptions for the symbols as in Fig. 2. All points represent an average (6S.E.M.) of two to six corneas. When no error bar is indicated, the standard error of the mean is smaller than the size of the symbol used.

stroma is hydrophilic. The epithelium and endothelium contain 100-fold greater amount of lipid material per unit weight than the stroma (Maurice and Mishima, 1984). Lipophilic drugs absorb into epithelium, but their partitioning from the epithelium to the stroma may become a penetration limiting factor (Mosher and Mikkelson, 1979). Consequently, adequate solubility both in epithelium and stroma is required. One way to achieve biphasic solubility is to have lipophilic prodrug that is converted to more hydrophilic derivative or parent drug in the epithelium (Suhonen et al., 1991a,b). Pilocarpine diester prodrugs are hydrolyzed to more hydrophilic monoesters and pilocarpine in the corneal epithelium, which has approximately twice the enzymatic activity per unit weight of the stroma and endothelium (Lee et al., 1982). Further hydrolysis of monoesters to pilocarpine takes place in the stroma and aqueous humor. In order to achieve adequate stability and solubility,

Fig. 5. Influence of the lipophilicity on the fraction of pilocarpine and the prodrugs absorbed into the cornea after 4 h perfusion. Descriptions for the symbols as in Fig. 2. All points represent an average (6S.E.M.) of two to six corneas. When no error bar is indicated, the standard error of the mean is smaller than the size of the symbol used.

pilocarpine and pilocarpine prodrugs must be formulated in an acidic eyedrop vehicle (Bundgaard et al., 1986b; Bundgaard and Hansen, 1982), which is neutralized after instillation on the ocular surface (Ahmed and Patton, 1984). In the case of pilocarpine (pK 9a 56.9) and its prodrugs (pK 9a 56.4–6.8) the ratios of un-ionized / ionized forms are, at pH 5.0, about 0.01 and 0.02–0.04 for pilocarpine and its prodrugs, respectively. In our study the permeability coefficient for pilocarpine was decreased more than three times when the pH value was decreased from 7.65 to 5.5. At the same pH range pilocarpine permeability when given as moderately lipophilic prodrugs I, III, and IV decreased 3, 11, and 14 times, respectively. For the more lipophilic II and V the pilocarpine permeability decreased only slightly with pH (Fig. 3). Due to the problems of detection and solubility, the pH range for V was only from 7.65 to 6.0. At acidic pH values the permeabilities of prodrugs in the cornea differed from pilocarpine less than they did at pH 7.65 and in some cases the permeabilities were lower than that of pilocarpine (Fig. 3). Thus, pH-dependent corneal permeability may have substantial effects on ocular bioavailability of pilocarpine prodrugs. It should be noted that ester prodrugs must be formulated generally at acidic pH. As corneal permeability of prodrugs increases substantially with increasing pH, the rate of neutralization on corneal surface should be an important determinant of their ocular absorption. The magnitude of these effects seems to be strongly dependent on the derivative (e.g. compare prodrugs IV and V) (Fig. 3). Despite its obvious importance there is no information available regarding the effects of pH on the permeation of lipophilic prodrugs through the cornea. According to the simple pH-partition hypothesis, only the un-ionized forms of drugs are able to pass through the cornea. The values of Pu and Pi from our experiments (Table 2) suggest the relative permeation rates of the un-ionized and ionized species change and the potential effect of pH. This is illustrated for pilocarpine and prodrugs in Fig. 2. Interestingly, the sigmoidal nature of the plot for IV was obtained with the inflection point near the pK 9a value 6.4, which suggests a possible pH-partition effect. Differences in corneal permeabilities of pilocarpine and its prodrugs are shown as a function of log (DC) in Fig. 6. Lowering the pH of donor solution from 7.65 to 4.50 caused a five-fold reduction in corneal uptake (Fig. 5) and seven-fold reduction in corneal permeability of pilocarpine (Fig. 3). Moreover, for I and II lowering the pH from 7.65 to 5.50 decreased the corneal uptake two-fold, while at the same time the corneal permeability decreased three and two fold, respectively. For compounds III and IV lowering the pH from 7.65 to 6.00 decreased the corneal uptake and permeability six-fold, but for compound IV lowering the pH further, to 5.00, decreased the corneal uptake 17-fold and at the same time the corneal permeability was decreased over 40 times. For lipophilic V lowering the pH to

P. Suhonen et al. / European Journal of Pharmaceutical Sciences 6 (1998) 169 – 176

Fig. 6. Corneal permeability coefficient of pilocarpine and the prodrugs versus log (DC). Descriptions for the symbols as in Fig. 2. All points represent an average (6S.E.M.) of two to six corneas. When no error bar is indicated, the standard error of the mean is smaller than the size of the symbol used.

6.00 neither affected its corneal uptake nor corneal permeability. These results suggest that partitioning of pilocarpine or prodrug from the donor buffer to the epithelium mainly determine the pH effects on permeability. Log (DC) of pilocarpine, I, and II was decreased about two units, when pH was decreased from 7.65 to 4.2. Log (DC) of O,O9-(1,4-xylylene) bispilocarpates III, IV, and V was decreased 4–5 units (Table 1). A large decrease in log (DC) as a function of pH may be due to two protonating imidazole groups in bispilocarpic derivatives compared to one imidazole in pilocarpine and pilocarpic acid diesters. Buffer pH did not affect the corneal integrity as permeation of mannitol and progesterone were not affected. We also have to consider that enzymatic activity in the cornea might be changed during 4 h study due to a downward shift, but the linear fluxes suggest that this is not the case. The pH of the epithelial cells is regulated by a Na 1 / H 1 exchanger (Aronson, 1985; Frelin et al., 1988; Grinstein et al., 1989; Moolenaar, 1986). From Fig. 5 it is obvious that the corneal uptake of bispilocarpic acid diesters seems to be smaller than the uptake of pilocarpic acid diesters, when we compare both groups at the same log (DC) value. This effect is quite clear at log (DC) range of 1.5–4.5. The smaller corneal uptake of O,O9-(1,4-xylylene) bispilocarpates compared to pilocarpic acid diesters is probably due to their larger molecular weights. In permeability smaller corneal uptake of bispilocarpic acid diesters is compensated when two pilocarpine molecules are released in the cornea from bispilocarpic acid diesters instead of one in the case of pilocarpic acid diesters. Analysis of samples from the epithelial side buffer solution showed both intact (bis)pilocarpic acid diester and pilocarpine. This could be due to either backdiffusion of pilocarpine from the cornea or leakage of esterases to the donor phase. In the control experiment, the cornea was incubated in the diffusion cell for 4 h, the epithelial side

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buffer solution was withdrawn and derivative IV was incubated in this solution. No hydrolysis of diester was detected during 4 h proving that no esterase leakage from the cornea took place. Thus, pilocarpine in the epithelial side buffer solution has diffused back from the cornea epithelium after cleavage from prodrugs. In the case of pilocarpine prodrugs, changes in the pH value also affect the formation of pilocarpine from the hydrolytically unstable (bis)pilocarpic acid monoesters (Schemes 1 and 2). Our HPLC-method did not allow measurement of bispilocarpic or pilocarpic acid monoesters from the endothelial side buffer. The degradation of (bis)pilocarpic acid monoesters are pH dependent being ¨ faster at neutral than acidic pH values (Jarvinen et al., 1992). The half-times of O,O9-(1,4-xylylene) bispilocarpate in aqueous solution at 378C are 18 min at pH 7.4, and ¨ 137 min at pH 5 (Jarvinen et al., 1992). For pilocarpic acid monoester the half-time in aqueous solution at pH 7.4 is 50 min (Bundgaard et al., 1986a). In order to confirm the hydrolytical degradation of (bis)pilocarpic acid monoesters to pilocarpine before HPLC analysis, the endothelial side samples were kept at room temperature before analysis. Consequently, the detected pilocarpine in the endothelial side buffer solution is the sum of penetrated pilocarpine and (bis)pilocarpic acid monoester. Also in vivo (bis)pilocarpic acid monoesters will be hydrolyzed to pilocarpine in the cornea and aqueous humor. Corneal uptake and subsequent penetration were sensitive to changes in solution pH values in the case of prodrugs with intermediate lipophilicity, whereas the pH effects were negligible for the most lipophilic compounds. These results have important implications in the design of eyedrop formulations from prodrugs. The data also demonstrates the limitations of the corneal in vitro permeability data at neutral pH and suggests the importance of eyedrop neutralization rate on ocular absorption in vivo.

Acknowledgements The authors thank Leiras (Finland), the Technology Development Centre (Finland) and Finnish Cultural ¨ Foundation for financial support. Ms. Paivi Perttula is acknowledged for her skilful technical assistance. Arto ¨ Urtti and Tomi Jarvinen are supported by Academy of Finland.

References Ahmed, I., Patton, T.F., 1984. Effect of pH and buffer on the precorneal disposition and ocular penetration of pilocarpine in rabbits. Int. J. Pharm. 19, 215–227. Anderson, R.A., Cowle, J.B., 1968. Influence of pH on the effect of pilocarpine on aqueous dynamics. Br. J. Ophthalmol. 52, 607–611. Aronson, P.S., 1985. Kinetic properties of the plasma membrane Na 1 –H 1 exchanger. Ann. Rev. Physiol. 47, 545–560.

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Bundgaard, H., Falch, E., Larsen, C., Mikkelson, T.J., 1986a. Pilocarpine prodrugs I. Synthesis, physicochemical properties and kinetics of lactonization of pilocarpic acid esters. J. Pharm. Sci. 75, 36–43. Bundgaard, H., Falch, E., Larsen, C., Mosher, G.L., Mikkelson, T.J., 1986b. Pilocarpine prodrugs II. Synthesis, stability, bioconversion, and physicochemical properties of sequentially labile pilocarpine acid diesters. J. Pharm. Sci. 75, 775–783. Bundgaard, H., Hansen, S.H., 1982. Hydrolysis and epimerization kinetics of pilocarpine in basic aqueous solution as determined by HPLC. Int. J. Pharm. 10, 281–289. Francoeur, M., Ahmed, I., Sitek, S., Patton, T.F., 1983. Age-related differences in ophthalmic drug disposition III. Corneal permeability of pilocarpine in rabbits. Int. J. Pharm. 16, 203–213. Francoeur, M.L., Sitek, S.J., Costello, B., Patton, T.F., 1985. Kinetic disposition and distribution of timolol in the rabbit eye. A physiologically based ocular model. Int. J. Pharm. 25, 275–292. Frelin, C., Vigne, P., Ladoux, A., Lazdunski, M., 1988. The regulation of the intracellular pH in cells from vertebrates. Eur. J. Biochem. 174, 3–14. Gibbs, I.S., Tuckerman, M.M., 1974. Formulation of a stable pilocarpine hydrochloride solution. J. Pharm. Sci. 63, 276–279. Grinstein, S., Rotin, D., Mason, M.J., 1989. Na 1 / H 1 exchange and growth factor-induced cytoplasmic pH changes. Role in cellular proliferation. Biochim. Biophys. Acta 988, 73–97. ¨ ¨¨ Jarvinen, T., Auriola, S., Peura, P., Suhonen, P., Urtti, A., Vepsalainen, J., 1991a. Synthesis and identification of pilocarpine acid diesters, prodrugs of pilocarpine. J. Pharm. Biomed. Anal. 9, 457–464. ¨ ¨¨ Jarvinen, T., Suhonen, P., Auriola, S., Vepsalainen, J., Urtti, A., Peura, P., 1991b. O,O9-(1,4-Xylylene) bispilocarpic acid esters as new potential double prodrugs of pilocarpine for improved ocular delivery. I. Synthesis and analysis. Int. J. Pharm. 75, 249–258. ¨ Jarvinen, T., Suhonen, P., Naumanen, H., Urtti, A., Peura, P., 1991c. Determination of physicochemical properties, stability in aqueous solutions and serum hydrolysis of pilocarpic acid diesters. J. Pharm. Biomed. Anal. 9, 737–745.

¨ Jarvinen, T., Suhonen, P., Urtti, A., Peura, P., 1991d. O,O9-(1,4-Xylylene) bispilocarpic acid esters as new potential double prodrugs of pilocarpine for improved ocular delivery. II. Physicochemical properties, stability, solubility and enzymatic hydrolysis. Int. J. Pharm. 75, 259– 269. ¨ Jarvinen, T., Suhonen, P., Urtti, A., Peura, P., 1992. Bispilocarpic acid monoesters as prodrugs of pilocarpine: II: physicochemical properties and kinetics of hydrolysis in aqueous solution. Int. J. Pharm. 79, 243–250. Lee, V.H.L., Morimoto, K.W., Stratford, R.E., 1982. Esterase distribution in the rabbit cornea and its implications in ocular drug bioavailability. Biopharm. Drug Disp. 3, 291–300. Maurice, D.M., Mishima, S., 1984. Pharmacology of the eye. In: Sears, M.L. (Ed.) Handbook of experimental pharmacology. Springer-Verlag, Berlin Heidelberg, pp. 16–119. Mitra, A.K., Mikkelson, T.J., 1988. Mechanism of transcorneal permeation of pilocarpine. J. Pharm. Sci. 77, 771–775. Moolenaar, W.H., 1986. Regulation of cytoplasmic pH by Na 1 / H 1 exchange. Trends Biochem. Sci. 11, 141–143. Mosher, G.L., Mikkelson, T.J., 1979. Permeability of the n-alkyl paminobenzoate esters across the isolated corneal membrane of the rabbit. Int. J. Pharm. 2, 239–243. Schoenwald, R.D., Huang, H.-S., 1983. Corneal penetration behavior of b-blocking agents I: physicochemical factors. J. Pharm. Sci. 72, 1266–1272. Sieg, J.W., Robinson, J.R., 1977. Vehicle effects on ocular drug bioavailability II: Evaluation of pilocarpine. J. Pharm. Sci. 66, 1222–1228. ¨ Suhonen, P., Jarvinen, T., Peura, P., Urtti, A., 1991a. Permeability of pilocarpic acid diesters across albino rabbit cornea in vitro. Int. J. Pharm. 74, 221–228. ¨ ¨ Suhonen, P., Jarvinen, T., Rytkonen, P., Peura, P., Urtti, A., 1991b. Improved corneal pilocarpine permeability with O,O9-(1,4-xylylene) bispilocarpic acid ester double prodrugs. Pharm. Res. 8, 1539–1542.