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Comparison of Enzymatic Hydrolysis of Pilocarpine Prodrugs in Human Plasma, Rabbit Cornea, and Butyrylcholinesterase Solutions
Comparison of Enzymatic Hydrolysis of Pilocarpine Prodrugs in Human Plasma, Rabbit Cornea, and Butyrylcholinesterase Solutions TOMIJARVINEN*~, MINNAPOIKOLAINEN*, PEKKASUHONEN~, JOUKO VEPSALAINENQ, SAKARIAMRANTAT,AND ARTOURTTI$ Received April 1, 1993, from the *Department of Pharmaceutical Chemistry and A. 1. Virtanen Institute, the #Department of Pharmaceutical Technology and A. I. Virtanen lnstitute, and the $Department of Chemistry, University of Kuopio, P. 0. Box 1627, FIN-70211, Accepted for publication November 21, 1994@. Kuopio, Finland, and YLeiras Oy, P. 0. Box 415, FlN-20101 Turku, Finland. -
Abstract 0 Various bispilocarpic acid diesters (double prodrugs of pilocarpine) were synthesized, and their in vitro esterase catalyzed hydrolysis was evaluated in diluted human plasma, rabbit cornea homogenate, and specific butyrylcholinesterase solution. The structural changes greatly affected the rate of enzymatic hydrolysis of the prodrugs. Bispilocarpic acid with 2 cyclopropane substituents was the most stable derivative, whereas bispilocarpic acid with 2 cyclobutane substituents was the most labile derivative. The charged bispilocarpic acid diester hydrolyzed more slowly than the uncharged form. Comparison of the results obtained from different plasma and cornea homogenate batches is difficult because of the variety of the enzyme systems involved. This variety also makes comparing the results between different laboratories difficult.
different laboratories is also very difficult because of the differences in the methods and the vehicle used. The specific esterases that are present in the eye are also a useful vehicle to study enzymatic hydrolysis of prodrug esters. By using specific esterases, it would be possible to study the effect of a defined enzyme, drug-protein binding could be avoided during the hydrolysis test, batch-to-batch variations of the vehicle would be avoided, and the results would be more comparable with other laboratories. The bispilocarpic acid diesters are promising double prodrugs of pilocarpine that have good aqueous solubility and stability, a wide range of lipophilicities, controllable rates of pilocarpine formation, and improved corneal penetration of the bispilocompared with p i l ~ c a r p i n e . ~ The ~.~ conversion ~ carpic acid diesters to pilocarpine is initiated by enzymatic hydrolysis t o the monoester, which is further hydrolyzed chemically t o pilocarpine. Previous studies of these diesters were carried out in human ~ 1 a s m a . l In ~ this study, we compared in vitro enzymatic hydrolysis of bispilocarpic acid diesters in a rabbit corneal homogenate, human plasma, and in butyrylcholinesterase solution.
Most reported ocular prodrugs are ester derivatives of the parent drug. The ocular prodrugs include esters of acyclovir,1,2 b-adrenoreceptor antagonist L-652.698,3carbonic anhydrase inhibitor^,^.^ epine~hrine,~,7 idoxuridine,8 nadol01,~ prostaglandins,1°-12 phenylephrine,13J4 pilocarpine,15-ls and t i m o l ~ l . ' ~Ester . ~ ~ derivatives can be used because most ophthalmic drugs contain hydroxyl or carboxyl groups that Experimental Section can be esterified to prodrugs with the desired lipophilicity, Chemicals-Pilocarpine hydrochloride was kindly provided by solution stability, and enzymatic lability. Ocular prodrugs are Leiras Oy (Tampere, Finland). Butyrylcholinesterase (BuChE, from designed t o release the parent drug enzymatically in the eye, horse serum) was purchased from Sigma Chemical Company (St. thereby allowing the drug to exert its effect. Louis, MO). All other chemicals were reagent or HPLC grade. In most cases, prodrugs release the parent drug in the S y n t h e s i s of Bispilocarpic Acid D i e s t e r s ( I - m - T h e cornea during absorption. Quantitative prediction of enzydetailed synthetic and identification procedures of bispilocarpic acid matic hydrolysis in vivo is, however, difficult because of the diesters I-IV (Figure 1)and XIII-XV (Figure 2) have been described complexities of the enzyme systems and partitioning and p r e v i o u ~ l y .The ~ ~ bispilocarpic ~~~ acid diesters V-XI1 (Figure 1)were synthesized according t o the previously described methods,31but the diffusion phenomena in the cornea and elsewhere in the eye. bispilocarpic acid diesters V,M,VIII, and M were isolated as a free The most commonly used ocular tissue in prodrug hydrolysis bases. The fumarate salts of these diesters were semisolid probably studies is the rabbit cornea,8,16,19,21,22 that contains true because of their low melting points and hygroscopicity. esterases, like a c e t y l c h o l i n e ~ t e r a s eand ~~~ butyrylcholinest~~ High-Resolution Mass Spectrometry-The positive-electronand carbonic a n h y d r a ~ ewhich , ~ ~ has some esterase impact ionization (EI) mass spectra of the bispilocarpic acid diesters activity.26 Total esterase activity (activity/mg protein) in V-MI were recorded on a VG 70-250SE magnetic sector mass human corneal epithelium and corneal stroma is 4.2- and 2.2spectrometer (VG Analytical, Manchester, U.K.) under the conditions fold, respectively, compared with that in corresponding albino described earlier.31.32 The accurate mass measurement of the morabbit tissues.27 The use of rabbit corneal and other ocular lecular ions was carried out under similar conditions, with perfluotissue homogenates is expensive and laborious. Consequently, rokerosene as a reference compound. human serum and plasma are oRen used in determination of NMR Spectroscopy-lH NMR spectra were recorded on a Bruker AM 400 the rates of enzymatic hydrolysis for ocular p r o d r ~ g s . ~ ~ J ~ *~ ~ WB , ~ ~spectrometer with a 5-mm 'HP3C-dual probe, operating a t 400.133 MHz for the 'H NMR measurements. To 0.6 mL of CD3Although plasma is a natural choice as a vehicle for evaluation OD, with Me&i (0.1%) as a n internal standard, was added 20-40 of the enzymatic hydrolysis of prodrugs for systemic adminmg of desired bispilocarpic acid diester. The number of data points istration, ocularly administered prodrugs should be hydroin the IH NMR experiment was 32 kW, with zerofilling to 128 kW, lyzed by the ocular tissues and not by plasma. In addition, total relaxation time of 16 s, 128 scans, and pulse angle of 90". enzyme distribution in the plasma and ocular tissues may be Liquid Chromatography-Liquid chromatography (LC) was different. In vitro enzymatic hydrolysis studies can also be performed with a system consisting of a Beckman programmable misleading because of interindividual variation in enzyme solvent module 116, a Beckman programmable UV-detector 166 (set systems. Variation is caused, for examples, genetic and a t 215 nm), a System Gold data module (Beckman Instruments Inc.), hormonal factors, sex, and age, which makes absolute rate and a Marathon autosampler (Spark Holland, AJ Emmen, The Netherlands), equipped with column thermostat and a Rheodyne predictions very difficult.29 Comparison of the results from 7080-080 loop (20 pL)injector. A deactivated Supelcosil LC8-DB (15 Abstract published in Advance ACS Abstracts, January 15, 1995.
656 /Journal of Pharmaceutical Sciences Vol. 84, No. 5, May 1995
cm x 4.6 mm id., 5 pm) reversed-phase column (Supelco, Bellefonte, PA) thermostated t o 40 "C was used as a stationary phase. The
0022-3549/95/3184-0656$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
t
-C
H
I
e CHz -
-CHI
I1
- CHICHI
111
-CH2CH2CH3
U
IV V
- CH~CHJ
- CHzCH,
-a
VI
-0
VII
vlll
.CH2CHI
CH2CHzCH2-
U -0
IX X
XI
- CH2CH2CH2CHZCH2 -
xn
- CH~CH~CH~CHZCHZCHZ -
U
Figure 1-Structures of the studied bispilocarpic acid diesters.
Compound
XI11
R2
CH, - CH, -
XIV
- CH, - CHz - CH,
xv
- CH2 - CHI - CH,
- CH, -
Figure 2-Structures of the studied bispilocarpic acid diesters. mobile phase was a mixture of methanol and 0.02 M KHzPOl (pH 4.5), which was individually optimized t o each compound. The flow rate was 1.0-1.2 mumin. In Vitro Enzymatic Rate of Hydrolysis in Human Plasma-The bispilocarpic acid diesters (2-5 ymol) were dissolved in 4.0 mL of phosphate buffer (0.16 M, pH 7.4, 37 "C). Then, 16.0 mL of pooled and preheated human plasma (Institute of Public Health, University of Kuopio) was added, and the solutions were kept in a water bath a t 37 "C. At suitable intervals, samples of 1.0 mL of the p1asma:buffer mixture were withdrawn and added to 3.0 mL of ethanol to deproteinize the plasma. ARer mixing and centrifugation, the supernatant was analyzed for remaining diester by the described LC method. The
rate constants for degradation were calculated from the linear regression slope of the logarithm of remaining bispilocarpic acid diesters versus time for several half-lives and normalized to the amount of proteins present in the incubation mixture. Coefficients of determination (1.2) for the plots were 0.98-1.00. In plasma hydrolysis experiments a t pH 6.0, the bispilocarpic acid diesters (1-lOpmol) were dissolved in the preheated (37 "C) mixture of p1asma:phosphate buffer (0.16 M; 80:20%, pH 6.0). Otherwise, the procedure was as described for pH 7.4. The protein concentration in the p1asma:buffer incubation solution (41 mg/mL) was determined by a dye-binding assay, with bovine serum albumin as the standard.33 I n Vitro E n z y m a t i c Rate of Hydrolysis in Rabbit C o r n e a Homogenate-The albino rabbits were sacrificed by pentobarbital sodium [intravenous (iv) injection, overdose]. The eyes were rinsed with 2.5 mL of 0.9% NaCl solution. The corneas were dissected and laid gently on the blotting paper for a few seconds and then deep frozen liquid N2. The corneas were stored a t -20 "C until used within 1month of collection. Twenty to fifty corneas were clipped into small pieces and placed into preweighed centrifuge tubes. The total weight of pieces was determined and cold tris buffer (0.05 M, pH 7.40) was added to give 20% (w/v) solutions. The corneas were then homogenized a t 4 "C with a MSE Soniprep 150 tissue homogenizer (MSE Scientific Instruments, Sussex, U.K.). The homogenate was subjected to centrifugation for 25 min a t 3500 rpm and 4 "C, yielding a clear supernatant. The supernatants were stored a t -20 "C until used. The fumarate salt of bispilocarpic acid diester (1.5 pmol) or free base of bispilocarpic acid diester (3.0 pmol) were dissolved into 25.0 and 50.0 mL of tris buffer (0.05 M, pH 7.401, respectively. Hydrolysis of the bispilocarpic acid diesters was studied by incubating 2000 pL of diester solution and 500 pL of corneal supernatant at 37 "C. At preselected times up t o 300 min, aliquots of 150 pL were withdrawn and mixed with 300 pL of ethanol to precipitate the soluble proteins and thereby terminate the reaction. The mixture was centrifuged, and 20 pL of clear supernatant was injected into the LC and analyzed for remaining diester by LC. The rate constants for degradation were calculated as they were in the case of the plasma studies, and the r2 values for the plots were 0.98-1.00. The protein concentration in the incubation solution varied between 0.44 and 0.51 mg/mL as determined by a dye-binding assay with bovine serum albumin a s the standard.33 I n Vitro Enzymatic Rate of Hydrolysis in Butyrylcholinesterase Solution-First, 2.5 pmol of the bispilocarpic acid diester was dissolved in 25.0 mL of preheated (37 "C) tris buffer (0.05M, pH 7.40). Butyrylcholinesterase (BuChE; 15 IU)was dissolved in 5.0 mL of preheated (37 "C) tris buffer (0.05 M, pH 7.40). A 5.0-mL sample of the diester (0.5 pmol) solution and a 5.0-mL sample of the enzyme solution (15 IU) were mixed, yielding a solution of 30 IU enzyme/ pmol substrate. The solution was kept in a waterbath a t 37 "C,and at preselected times up to 300 min, 200 pL of the contents of the vial were withdrawn and mixed with 400 pL of ethanol to precipitate the proteins and terminate the reaction. The mixture was centrifuged, and 20 pL of clear supernatant was injected into the LC and was analyzed for remaining diester by the same LC method. The rate constants for degradation were calculated as before, and the r2 values for the plots were 0.98-1.00. M e a s u r e m e n t of A p p a r e n t Partition Coefficient-The lipophilicities of the bispilocarpic acid diesters were obtained from the distribution of the compounds between 1-octanol and phosphate buffer.34 The apparent partition coefficients were determined a t pH 5.0 to avoid solubility problems a t pH 7.4. The phosphate buffer (0.16 M, pH 5.0, p = 0.5 adjusted with NaC1) and 1-octanol phases were saturated before use by stirring vigorously for 24 h a t room temperature. The determinations of prodrugs concentrations and the calculations of apparent partition coefficients were carried out as described previously.17
Results and Discussion Identification of Bispilocarpic Acid Diesters-The most prominent fragment ions with respect to relative intensity of the bispilocarpic acid diesters V-XI1 are listed in Table 1. All the mass spectra show a molecular ion [M+] with low intensity and display corresponding fragment ions like the bispilocarpic acid diesters described earlier.31 The elemental Journal of Pharmaceutical Sciences / 657 Vol. 84, No. 5, May 1995
Table 1-Relative Abundances of the Ions in the El Mass Spectra of the Bispilocarpic Acid Diesters V - XI1 Compound
[M+.]
Other Fragment Ions
V VI VII Vlll
590 (5%) 614 (1%) 642(4%) 604 (6%)
IX
628 (7%)
X XI XI1
656 (6%) 656 (3%) 670 (2%)
503 (8%),397 (19%),396 (85%),382 (20%),381 (95%),309 (llyo),209 (18%), 195 (45%),96 (78%),95 (100%) 408 (11O/0),393 (lo%),320 (29%),291 (l6%),209 (l8Y0),208 (llYo),207 (78%),163 (29%),121 (47%),113 (52%),96 (38%),95 (100%). 436 (lo%), 423 (22%),421 (loo%), 421 (13%),408 (18%), 407 (85%),335 (lo%),221 (52%),209 (12%)' 121 (13%),96 (86%),95 (50%). 517 (7%),411 (13%),410 (57%),396 (16%),395 (71%),339 (36%),323 YO), 209 (IS%),208 (6%),195 (48%),121 (20%),96 (56%), 95 (100%). > - - -, 423(19%),422 (76%),408 (16%),407 (73%),352 (lo%), 351 (40%),334 (lo%),209 (19%),208 (1 l%),207 (73%),163 (21%),121 (32%), 96 (55%).95 (100%). 543 (9%),437 (23%),436 (loo%),422 (22%),421 (86%),366 (lo%),365 (46%),221 (58%),209 (14%),121 (15%),96 (68%),95 (78%) 450 (39%),435 (23%),379 (13%),363 (13%),209 (15%),207 (29%),163 (ll%),121 (23%),96 (50%),95 (100%). 464 (13),449 (lo%), 377 (lo%),209 (ll%),207 (21%),163 (l6%),121 (26%),96 (53%),95 (100%).
Table 2-Measured and Calculated Accurate Mass of Bispilocarpic Acid Diesters ~~~
Compound
Observed Mass
Calculated Mass
Error (mmu)
V VI VII Vlll IX X XI XI1
590.3363040 614.3299260 642.3648680 604.3491210 628.3428340 656.3743290 656.3775330 670.3941500
590.3315650 614.3315650 642.3628652 604.3472151 628.3472151 656.3785152 656.3785152 670.3941653
4.7 1.6 2.0 1.9 4.4 4.2 1.o 0.0
~
Elemental composition (free base)
Table 3-Proton Chemical Shifts for Compounds V-XI1 Chemical Shift (ppm) Proton
V
VI
VII
Vlll
IX
X
Xi
XI1
N-Me 2 4 6a 6b 7 8 9 10 11
3.61 7.51 6.73 2.67 2.67 2.31 2.50 1.67 0.90 4.08 4.34
3.63 7.58 6.78 2.69 2.69 2.33 2.50 1.69 0.92 4.08 4.35 1.61 0.90 0.90 -
3.79 8.50 7.23 2.77 2.77 2.40 2.54 1.71 0.93 4.11 4.36 3.16 2.22 2.01 1.89 6.73
3.62 7.56 6.76 2.69 2.69 2.33 2.49 1.68 0.91 4.08 4.18 2.01 2.31 1.10 1.10 -
3.61 7.51 6.73 2.68 2.68 2.33 2.49 1.68 0.91 4.07 4.19 2.02 1.62 0.90 0.90 -
3.80 8.50 7.23 2.78 2.78 2.40 2.53 1.71 0.92 4.11 4.21 2.03
3.77 8.34 7.15 2.77 2.77 2.38 2.51 1.69 0.91 4.09 4.14 1.72 1.48 1.61 0.88 0.88
3.81 8.53 7.24 2.79 2.79 2.38 2.52 1.69 0.91 4.09 4.12 1.69 1.45 1.61 0.90 0.90 6.72
Riy
R2a R$ R$' R2Y Fum. a
-a
2.32 1.10 1.10 -
-
-
-
3.16 2.22 2.01 1.89 6.73
-
6.71
-, Not applicable.
composition of the molecular ion was determined by measuring the accurate mass of the molecular ion. The error between observed and calculated mass <4.7 mmu for diesters V-XI1 (Table 21, which reliably verifies the elemental composition of the bispilocarpic acid diesters. The NMR investigation, together with mass spectrometry, ensures correct identification of the new drug substances. The IH NMR data of the bispilocarpic acid diesters V-XI1 were consistent with their structure. IH assignments were based on the 'H-'H-correlated spectra (COSY) and are listed in Table 3. Enzymatic Hydrolysis of Bispilocarpic Acid D i e s t e r s in Different Vehicles-Bispilocarpic acid diesters are very stable towards chemical hydrolysis in the absence of enz y m e ~ . ' However, ~ in the presence of esterases, prodrugs will be hydrolyzed to the corresponding monoesters with a char658 /Journal of Pharmaceutical Sciences Vol. 84, No. 5, May 1995
Rate Constant ( h - h g protein)
Log P a t
~
Rla RIB
Table 4-Apparent Partition Coefficient at pH 5.0 and Rates of Hydrolysis of Various Bispilocarpic Acid Diesters in 80% Human Plasma (pH 7.40), Homogenates of the Cornea of the Albino Rabbit (pH 7.40), and Butyrylcholinesterase Enzyme Solution (pH 7.40) at 37 "C
Prodrug
pH 5.0 (n = 3)
I II 111 IV V VI VII Vlll IX X XI XI1
-0.29 i 0.01 0.66 ? 0.05 1.69f 0.01 1.03i0.07 -0.77 i 0.04 -0.58 i0.01 0.30 i 0.01 -0.61 i 0.01 -0.30 i0.03 0.60 i 0.01 0.40 f 0.01 0.88 f 0.02
80% Human Plasma (n = 3)
Rabbit Cornea Butylylcholinesterase Homogenate Enzyme Solution (n = 2-4) (n = 3)
0.0076 f 0.00115 2.2 ?k 0.28 0.0086 i 0.00018 10.5 f 0.34 0.0055i0.00067 12.6 i2.05 0.0037 i- 0.00006 2.2 i 0.13 0.0166 L 0.00056 2.7 i 0.39 1.3 i 0.03 0.0016 f 0.00027 0.0081 f 0.00044 9.3 i 1.62 3.6 i 0.42 0.0093 f 0.00008 0.0008 i 0.00001 2.3 i 0.35 0.0094 f 0.00061 10.6i 1.13 0.0037 L 0.00007 1.7 i 0.19 0.0036 i 0.00031 2.5 i 0.03
1.3 2 0.05 8.1 f 0.04 15.1 f 2.24 1.8 f 0.06 5.1 i 0.68 0.8 f 0.01 15.4 f 0.39 4.5 f 0.17 0.5 f 0.02 14.5 f 2.52 0.9 i 0.05 0.7 i 0.02
NOTE: Results are expressed as mean f SE.
aderistic rate. The rate constants (normalized to the amount of proteins in t h e incubation mixture) for the hydrolysis of bispilocarpic acid diesters in diluted human plasma, rabbit cornea homogenate, and BuChE solution are listed in Table 4. The true comparison of rate constant of the same compound in different vehicles is not meaningful because the total activity of esterases and the maximum velocity (Vmm)and Michaelis constant (K,) values of diesters may vary in these systems. The substrate concentration in diluted human plasma varied from 100 to 250 pM (pH 7.4) and from 50 to 500 pM (pH 6.0) depending on the solubility and UV absorption of the prodrugs. At low substrate concentrations ([S] < 0.1 Km),the absolute rate of enzymatic hydrolysis is linearly dependent on the substrate concentration and the reaction follows firstorder kinetics. In this case, half-time is independent of the substrate concentration. The K , value was calculated to be 1.6 mM (pH 7.4,37 "C) for derivative I1from the LineweaverBurk plot (Figure 3), and the half-life (~-112) was independent of the substrate concentration in plasma hydrolysis. The K, was not determined for hydrolysis in cornea homogenate and i n BuChE solution. To confirm t h a t substrate concentration does not affect the t l l z of the diester, the same substrate concentration was used in every hydrolysis determinations in cornea homogenate and BuChE solution for each prodrug. The effect of substrate concentration on the rate of hydrolysis i n BuChE solution is shown in Table 5 . This result confirms that if the relationship [prodrugl/[enzymel is not small enough, the substrate concentration affects the value of tllz and thus the comparisons of the tllz values obtained from different laboratories are not reasonable.
y = 0 21003 + 3 4 1 . 4 1 ~ R ^ 2
=
Table 6-Half-Lives of Selected Bispilocarpic Acid Diesters in 80% Human Plasma Solution (pH 6.0) at 37 "C
0 990
f112 (min)
t112
Compd
Plasma (Batch A)
Plasma (Batch B)a
I I1 111 IV
13 5 4 26
46 42 19 269
a
Compd Xlll XIV
xv
(min)
Plasma (Batch A)
Plasma (Batch B)d
34 1 7 3
735 46 10
Plasma batch B is similar to plasma used in experiments at pH 7.4 in Table
4.
0 005
0 000
0010
0005
0 L15
0020
I/ISI tpM-')
Figure 3-Lineweaver-Burk plot of l/vo versus l/[S] for prodrug It initial velocity of enzymatic hydrolysis at diester concentration [q).
(yo
= the
Table 5-Effect of Relation BuChE Activity to the Half-Time of Derivative II BuChE Activity (luipmol substrate) 3
9 15
hi2
(min)
Degradation was not observed 52 23
BuChE Activity (IUlpmol substrate)
tIi2(min)
15
23
27 59
10 3.5
The derivatives 11, 111, V, VII, VIII, and X are good substrates for BuChE and they are also hydrolyzed rapidly in human plasma and rabbit cornea homogenate (Table 4). Consequently, these prodrugs will be hydrolyzed primarily by BuChE also in the cornea and plasma. The rate of hydrolysis by BuChE increased with increasing chain length of acyl substituent a t Rz (I, 11, 111). BuChE is the dominant esterase in rabbit ocular tissues.27 For example PGFzu esters are hydrolyzed by BuChE but not by acetylcho. ~ ~ naphthyl esters with linesterase or carbonic a n h y d r a ~ e The ester chains longer than four carbons are primarily hydrolyzed by BuChE, and for 1- and 2-naphthyl esters, the maximal hydrolytic rates by BuChE were reached at valerate (C5) and at butyrate (C4) esters, r e s p e c t i ~ e l y .A ~ ~similar parabolic chain length dependence of hydrolysis of 1- and 2-naphthyl esters in rabbit ocular tissue has also been reported.36 Thus, the ocular prodrug should preferably have a butyryl portion as an acyl group t o hydrolyze rapidly by BuChE in the eye. On the other hand, optimal prodrug ester should be hydrolyzed by several esterases to minimize the effect of individual fluctuations in the proportion of esterases in the eye.37 The results of the hydrolysis studies show that the rate of enzymatic hydrolysis is greatly dependent on the ester structure in every vehicle. The most stable derivatives in this series are the compounds that have the cyclopropyl as Rz (IV, VI,M,XI, and XII). This result is in good agreement with other e n z y m a t i ~and ~ ~c~h~e ~m i ~ a lhydrolysis ~ ~ , ~ ~ studies of prodrugs. The stability of the cyclopropanoyl ester is due to steric effects of the cyclic side chain.39 It is evident that increased branching of the alkyl group in the acyl portion of the prodrug esters (e.g., pivaloyl and isopropyl esters) decrease
the rate of enzymatic The most labile derivatives in this series for enzymatic hydrolysis are 0,O'dicyclobutane derivatives (Table 4; VII, X), which may be due t o the flexibility of the cyclobutane structure that may decrease the steric hindrance to attack by esterases. Although the RZ moiety of bispilocarpic acid diesters controls the rate of enzymatic conversion to the corresponding bispilocarpic acid monoesters, and the structure of the R1 determines the rate of chemical hydrolysis t o p i l o ~ a r p i n e , ~ ~ ~ ~ variations in the R1 moiety may also have an effect on the rate of enzymatic hydrolysis (Table 4). For example, prodrugs IV,VI, IX,XI, and XI1 have the same Rz, but R1 is varied; this change in R1 causes variations in the rate of enzymatic hydrolysis, especially in BuChE solution and in diluted human plasma. Thus, both pro-moieties of double prodrugs may have a n effect on the reaction that affects only the other pro-group. This conclusion is not in agreement with the studies on corresponding pilocarpic acid diesters.l6Yz1 Possibly the R1 group has a more remarkable effect on the conformation of bis-structure than on mono-structure. On the other hand, in prodrugs V, VIII, VII, and X, R2 is similar but Rl is varied and the rates of enzymatic hydrolysis were similar. Thus, the effect of R1 structure on the rate of enzymatic hydrolysis of R2 is also dependent on the structure of Rz. Effect of pH on Enzymatic Hydrolysis in Human Plasma-The bispilocarpic acid diesters are very lipophilic weak bases (pK, x 7)17J8that are poorly water soluble at pH 7.4. Because of this poor water solubility, hydrolysis studies a t pH 7.4 with HPLC determination suffer from sensitivity problems. We studied enzymatic hydrolysis at pH 6.0 in diluted human plasma to decrease the solubility problems. The rate of enzymatic hydrolysis a t pH 6.0 was clearly slower than the corresponding value at pH 7.4; therefore, it is not reasonable to use pH 6.0 instead of 7.4. This slow rate of hydrolysis may be due to the protonation of prodrugs, which leads to structural and electrostatic changes.43 This slow rate of hydrolysis at pH 6.0 may also suggests that whereas the uncharged form of the bispilocarpic acid diesters is hydrolyzed enzymatically the protonated form is not. The slight decrease of cholinesterase activity at pH 6.044may have only a minor effect on the rate of enzymatic hydrolysis. During the enzymatic hydrolysis studies at pH 6.0, we found that the results can vary widely depending on plasma batch even though we used pooled plasma. The half-lives of enzymatic hydrolysis of some selected derivatives in two different plasma batches are presented in Table 6. Major variations in plasma may be due to different patient groups. Although the final explanation was not found, this result indicates that the whole analogy series must be studied in the same plasma or serum batch and the results from different laboratories can be dependent on the enzyme systems used. Related problems are involved when eye homogenate is used because the esterase activity in rabbit eye changes with the tissue,45 age,46s47and p i g m e n t a t i ~ n . ~ ~ Effect of Lipophilicity of the Prodrug on Enzymatic Hydrolysis-All bispilocarpic acid diesters are more lipophilic Journal of Pharmaceutical Sciences / 659 Vol. 84, No. 5, May 1995
than the parent compound, pilocarpine (log P = -1.72 at pH 5.01,mainly because of replacement of the lactone ring by ester groups (Table 4). Opening and esterification of the lactone ring have no effect on the ionization of the imidazole moiety of bispilocarpic a ~ i d , ~and, ~ . ' thus, ~ the observed changes in lipophilicity are not due to the decrease in t h e pK,. It has also been shown that the drug-protein binding generally increases with increasing partition coefficient of a For example, protein binding of pilocarpic acid monoesters increases with increasing partition coefficient.21 Thus, it is possible that the rates of enzymatic hydrolysis of lipophilic prodrugs become slower because of strong protein binding especially if the in uitro experiments have been done in vehicles with high protein concentration (plasma, serum). The effect of protein binding on enzymatic hydrolysis is difficult to clarify by determining the rate of hydrolysis K,) of substrates in because all other properties (e.g., V, whole series should be constant. However, the results in Table 4 show that increased lipophilicity of bispilocarpic acid diesters and possibly related protein binding do not have clear effects on the enzymatic hydrolysis.
References and Notes 1. Maudgal, P. C.; Clercq, K. D.; Descamps, J.; Missotten, L. Arch. Ophthalmol. 1984, 102, 140-142. 2. Bundgaard, H.; Jensen, E.; Falch, E. Pharm. Res. 1991,8,10871----. nw 3. Sugrue, M. F.; Gautheron, P.; Grove, J.; Mallorga, P.; Viader, M.-P.; Baldwin, J . J.; Ponticello, G. S.; Varga, S. L. Inuest. Ophthalmol. Vis. Sci. 1988, 29, 776-784. 4. Grove, J.; Gautheron, P.; Plazonnet, B.; Sugrue, M. F. J . Ocul. Pharmacol. 1988,4, 279-290. 5. S u m e . M. F.: Gautheron. P.: Scmitt. C.: Viader. M. P.: Conauet. R . 1 . ; Smith,'R. L.; Share, N. N.; Stone, C. A. J . Pharmhcol: Exp. Ther. 1985,232, 534-540. 6. Mandell, A. I.; Stentz, F.; Kitabchi, A. E. Ophthalmology 1978, 85, 268-275. 7. Kaback. M. B.: Podos. S. M.: Harbin. T. S.: Mandell. A.: Becker. B. A m . J. Ophthalmol. 1976, 81, 768-772. 8. Narurkar, M. M.; Mitra, A. K. Pharm. Res. 1989, 6, 887-891. 9. Duzman, E.; Chen, C.-C.; Anderson, J.; Blumenthal, M.; Twizer, H. Arch. Ophthalmol. 1982,100, 1916-1919. 10. Villumsen, J.; Alm, A,; Soderstrom, M. Br. J . Ophthalmol. 1989, 73,975-979. 11. Villumsen, J.; Alm, A. Br. J . Ophthalmol. 1989, 73, 419-426. 12. Bito, L. Z.; Baroody, R. A. Exp. Eye Res. 1987,44, 217-226. 13. Mindel, J. S.; Shaikewitz, S. T.; Podos, S. M. Arch. Ophthalmol. 1980, 98, 2220-2223. 14. Yuan. S.-S.: Bodor. N. J . Pharm. Sci. 1976. 65. 929-931. 15. Bundgaard,'H.; Falch, E.; Larsen, C.; Mosher, G. L.; Mikkelson, T. J . J . Pharm. Sci. 1986, 75, 36-43. 16. Bundgaard, H.; Falch, E.; Larsen, C.; Mosher, G. L.; Mikkelson, T. J. J . Pharm. Sci. 1986, 75, 775-783. 17. Jarvinen, T.: Suhonen., P.:, Urtti. A.: Peura. P. Int. J. Pharm. 1991, 75, 259-269. 18. Jarvinen, T.; Suhonen, P.; Launonen, M.; Peura, P.; Urtti, A. S. T . P. Pharm. Sci. 1992,2, 53-60. I
I
660 / Journal of Pharmaceutical Sciences Vo/. 84, No. 5, May 1995
,
,
19. Bundgaard, H.; Buur, A,; Chang, S.-C.; Lee, V. H. L. Int. J . Pharm. 1986,33, 15-26. 20. Bundgaard, H.; Buur, A,; Chang, S.-C.; Lee, V. H. L. Znt. J . Pharm. 1988,46, 77-88. 21. Mosher, G. L. Ph.D. Thesis, University of Kansas, 1986. 22. Camber, 0.; Edman, P. Int. J . Pharm. 1987, 37, 27-32. 23. Petersen, R. A.; Lee, K.-J.; Donn, A. Arch. Ophthalmol. 1965, 73. 370-377. 24. Lee, V. H. L.; Chang, S.-C.; Oshiro, C. M.; Smith, R. E. Curr. E.ye Res. 1985, 4 , 1117-1125. 25. Lonnerholm. G. Acta Phvsiol. Scand. 1974. 90. 143-152.
28. Bodor, N.; Visor, G. Pherm. Res. 1984,168-173. 29. Sinkula, A. A,; Yalkowsky, S. H. J . Pharm. Sci. 1975,64, 181733. 30. Suhonen, P.; Jarvinen, T.; Rytkonen, P.; Peura, P.; Urtti, A. Pharm. Res. 1991,8, 1539-1542. 31. Jarvinen, T.; Suhonen, P.; Auriola, S.; Vepsalainen, J.; Urtti, A.; Peura, P. Znt. J . Pharm. 1991, 75, 249-250. 32. Jarvinen, T.; Suhonen, P.; Auriola, S.; Vepsalainen, J.; Urtti, A,; Peura, P. J . Pharm. Biomed.Ana1. 1992, 10, 153-161. 33. Bradford, M. M. Anal. Chem. 1976, 72, 248-254. 34. Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71,525-616. 35. Camber, 0.;Edman, P.; Olsson, L.-I. Znt. J . Pharm. 1986,29, 259-266. 36. Chang, S.-C.; Lee, V. H. L. Curr. Eye Res. 1983,2, 651-656. 37. Lee, V. H. L.: Li. V. H. K. Adu. Drug. Del. Rev. 1989. 3. 1-38. 38. Chien, D.-S.;Sasaki, H.; BundgaardrH.; Buur, A.; Lee, V. H. L. Pharm. Res. 1991,8, 728-733. 39. Geraldine, C.; Jordan, M.; Quigley, J . M.; Timoney, R. F. Int. J . Pharm. 1992.84. 175-189. 40. Kristoffersson, J.5 Svensson, L. A.; Tegner, K. Acta Pharm. Suec. 1974,11, 427-438. 41. Huang, H. P.; Ayres, J. W. J . Pharm. Sci. 1988, 77, 104-109. 42. Jarvinen, T.; Suhonen, P.; Urtti, A.; Peura, P. Int. J . Pharm. 1991, 79, 243-250. 43. Konschin, H.; Ekholm, M. Znt. J . Quantum Chem., Quantum Biol. Symp. 1991,18, 247-267. 44. Lehringer, L. Biochemistry; Worth Publishers, Inc., New York, 1975; p 196. 45. Lee, V: H. L.; Morimoto, K. W.; Stratford, R. E. Biopharm. Drug. Dispos. 1982, 3, 291-300. 46. Lee, V. H. L.; Stratford, R. E.; Morimoto, K. W. Znt. J . Pharm. 1983.13, 183-195. 47. Redell, M. A.; Yang, D. C.; Lee, V. H. L. Znt. J . Pharm. 1983, 17, 299-312. 48. Lee, V. H. L. J . Pharm. Sci. 1983, 72, 239-244. 49. Kakemi, K.; Arita, T.; Hori, R.; Konishi, R.; Nishimura, K. Chem. Pharm. Bull. 1969,17, 248-255.
Acknowledgments This study was supported by a grant from Leiras Oy (Finland) and the Technology Development Centre (Finland). Art0 Urtti was supported by the Academy of Finland. Ms. Paivi Varis and Mrs. Marja Mali is gratefully acknowledged for her skillful technical assistance.
JS930091G
Abstract 0 Various bispilocarpic acid diesters (double prodrugs of pilocarpine) were synthesized, and their in vitro esterase catalyzed hydrolysis was evaluated in diluted human plasma, rabbit cornea homogenate, and specific butyrylcholinesterase solution. The structural changes greatly affected the rate of enzymatic hydrolysis of the prodrugs. Bispilocarpic acid with 2 cyclopropane substituents was the most stable derivative, whereas bispilocarpic acid with 2 cyclobutane substituents was the most labile derivative. The charged bispilocarpic acid diester hydrolyzed more slowly than the uncharged form. Comparison of the results obtained from different plasma and cornea homogenate batches is difficult because of the variety of the enzyme systems involved. This variety also makes comparing the results between different laboratories difficult.
different laboratories is also very difficult because of the differences in the methods and the vehicle used. The specific esterases that are present in the eye are also a useful vehicle to study enzymatic hydrolysis of prodrug esters. By using specific esterases, it would be possible to study the effect of a defined enzyme, drug-protein binding could be avoided during the hydrolysis test, batch-to-batch variations of the vehicle would be avoided, and the results would be more comparable with other laboratories. The bispilocarpic acid diesters are promising double prodrugs of pilocarpine that have good aqueous solubility and stability, a wide range of lipophilicities, controllable rates of pilocarpine formation, and improved corneal penetration of the bispilocompared with p i l ~ c a r p i n e . ~ The ~.~ conversion ~ carpic acid diesters to pilocarpine is initiated by enzymatic hydrolysis t o the monoester, which is further hydrolyzed chemically t o pilocarpine. Previous studies of these diesters were carried out in human ~ 1 a s m a . l In ~ this study, we compared in vitro enzymatic hydrolysis of bispilocarpic acid diesters in a rabbit corneal homogenate, human plasma, and in butyrylcholinesterase solution.
Most reported ocular prodrugs are ester derivatives of the parent drug. The ocular prodrugs include esters of acyclovir,1,2 b-adrenoreceptor antagonist L-652.698,3carbonic anhydrase inhibitor^,^.^ epine~hrine,~,7 idoxuridine,8 nadol01,~ prostaglandins,1°-12 phenylephrine,13J4 pilocarpine,15-ls and t i m o l ~ l . ' ~Ester . ~ ~ derivatives can be used because most ophthalmic drugs contain hydroxyl or carboxyl groups that Experimental Section can be esterified to prodrugs with the desired lipophilicity, Chemicals-Pilocarpine hydrochloride was kindly provided by solution stability, and enzymatic lability. Ocular prodrugs are Leiras Oy (Tampere, Finland). Butyrylcholinesterase (BuChE, from designed t o release the parent drug enzymatically in the eye, horse serum) was purchased from Sigma Chemical Company (St. thereby allowing the drug to exert its effect. Louis, MO). All other chemicals were reagent or HPLC grade. In most cases, prodrugs release the parent drug in the S y n t h e s i s of Bispilocarpic Acid D i e s t e r s ( I - m - T h e cornea during absorption. Quantitative prediction of enzydetailed synthetic and identification procedures of bispilocarpic acid matic hydrolysis in vivo is, however, difficult because of the diesters I-IV (Figure 1)and XIII-XV (Figure 2) have been described complexities of the enzyme systems and partitioning and p r e v i o u ~ l y .The ~ ~ bispilocarpic ~~~ acid diesters V-XI1 (Figure 1)were synthesized according t o the previously described methods,31but the diffusion phenomena in the cornea and elsewhere in the eye. bispilocarpic acid diesters V,M,VIII, and M were isolated as a free The most commonly used ocular tissue in prodrug hydrolysis bases. The fumarate salts of these diesters were semisolid probably studies is the rabbit cornea,8,16,19,21,22 that contains true because of their low melting points and hygroscopicity. esterases, like a c e t y l c h o l i n e ~ t e r a s eand ~~~ butyrylcholinest~~ High-Resolution Mass Spectrometry-The positive-electronand carbonic a n h y d r a ~ ewhich , ~ ~ has some esterase impact ionization (EI) mass spectra of the bispilocarpic acid diesters activity.26 Total esterase activity (activity/mg protein) in V-MI were recorded on a VG 70-250SE magnetic sector mass human corneal epithelium and corneal stroma is 4.2- and 2.2spectrometer (VG Analytical, Manchester, U.K.) under the conditions fold, respectively, compared with that in corresponding albino described earlier.31.32 The accurate mass measurement of the morabbit tissues.27 The use of rabbit corneal and other ocular lecular ions was carried out under similar conditions, with perfluotissue homogenates is expensive and laborious. Consequently, rokerosene as a reference compound. human serum and plasma are oRen used in determination of NMR Spectroscopy-lH NMR spectra were recorded on a Bruker AM 400 the rates of enzymatic hydrolysis for ocular p r o d r ~ g s . ~ ~ J ~ *~ ~ WB , ~ ~spectrometer with a 5-mm 'HP3C-dual probe, operating a t 400.133 MHz for the 'H NMR measurements. To 0.6 mL of CD3Although plasma is a natural choice as a vehicle for evaluation OD, with Me&i (0.1%) as a n internal standard, was added 20-40 of the enzymatic hydrolysis of prodrugs for systemic adminmg of desired bispilocarpic acid diester. The number of data points istration, ocularly administered prodrugs should be hydroin the IH NMR experiment was 32 kW, with zerofilling to 128 kW, lyzed by the ocular tissues and not by plasma. In addition, total relaxation time of 16 s, 128 scans, and pulse angle of 90". enzyme distribution in the plasma and ocular tissues may be Liquid Chromatography-Liquid chromatography (LC) was different. In vitro enzymatic hydrolysis studies can also be performed with a system consisting of a Beckman programmable misleading because of interindividual variation in enzyme solvent module 116, a Beckman programmable UV-detector 166 (set systems. Variation is caused, for examples, genetic and a t 215 nm), a System Gold data module (Beckman Instruments Inc.), hormonal factors, sex, and age, which makes absolute rate and a Marathon autosampler (Spark Holland, AJ Emmen, The Netherlands), equipped with column thermostat and a Rheodyne predictions very difficult.29 Comparison of the results from 7080-080 loop (20 pL)injector. A deactivated Supelcosil LC8-DB (15 Abstract published in Advance ACS Abstracts, January 15, 1995.
656 /Journal of Pharmaceutical Sciences Vol. 84, No. 5, May 1995
cm x 4.6 mm id., 5 pm) reversed-phase column (Supelco, Bellefonte, PA) thermostated t o 40 "C was used as a stationary phase. The
0022-3549/95/3184-0656$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
t
-C
H
I
e CHz -
-CHI
I1
- CHICHI
111
-CH2CH2CH3
U
IV V
- CH~CHJ
- CHzCH,
-a
VI
-0
VII
vlll
.CH2CHI
CH2CHzCH2-
U -0
IX X
XI
- CH2CH2CH2CHZCH2 -
xn
- CH~CH~CH~CHZCHZCHZ -
U
Figure 1-Structures of the studied bispilocarpic acid diesters.
Compound
XI11
R2
CH, - CH, -
XIV
- CH, - CHz - CH,
xv
- CH2 - CHI - CH,
- CH, -
Figure 2-Structures of the studied bispilocarpic acid diesters. mobile phase was a mixture of methanol and 0.02 M KHzPOl (pH 4.5), which was individually optimized t o each compound. The flow rate was 1.0-1.2 mumin. In Vitro Enzymatic Rate of Hydrolysis in Human Plasma-The bispilocarpic acid diesters (2-5 ymol) were dissolved in 4.0 mL of phosphate buffer (0.16 M, pH 7.4, 37 "C). Then, 16.0 mL of pooled and preheated human plasma (Institute of Public Health, University of Kuopio) was added, and the solutions were kept in a water bath a t 37 "C. At suitable intervals, samples of 1.0 mL of the p1asma:buffer mixture were withdrawn and added to 3.0 mL of ethanol to deproteinize the plasma. ARer mixing and centrifugation, the supernatant was analyzed for remaining diester by the described LC method. The
rate constants for degradation were calculated from the linear regression slope of the logarithm of remaining bispilocarpic acid diesters versus time for several half-lives and normalized to the amount of proteins present in the incubation mixture. Coefficients of determination (1.2) for the plots were 0.98-1.00. In plasma hydrolysis experiments a t pH 6.0, the bispilocarpic acid diesters (1-lOpmol) were dissolved in the preheated (37 "C) mixture of p1asma:phosphate buffer (0.16 M; 80:20%, pH 6.0). Otherwise, the procedure was as described for pH 7.4. The protein concentration in the p1asma:buffer incubation solution (41 mg/mL) was determined by a dye-binding assay, with bovine serum albumin as the standard.33 I n Vitro E n z y m a t i c Rate of Hydrolysis in Rabbit C o r n e a Homogenate-The albino rabbits were sacrificed by pentobarbital sodium [intravenous (iv) injection, overdose]. The eyes were rinsed with 2.5 mL of 0.9% NaCl solution. The corneas were dissected and laid gently on the blotting paper for a few seconds and then deep frozen liquid N2. The corneas were stored a t -20 "C until used within 1month of collection. Twenty to fifty corneas were clipped into small pieces and placed into preweighed centrifuge tubes. The total weight of pieces was determined and cold tris buffer (0.05 M, pH 7.40) was added to give 20% (w/v) solutions. The corneas were then homogenized a t 4 "C with a MSE Soniprep 150 tissue homogenizer (MSE Scientific Instruments, Sussex, U.K.). The homogenate was subjected to centrifugation for 25 min a t 3500 rpm and 4 "C, yielding a clear supernatant. The supernatants were stored a t -20 "C until used. The fumarate salt of bispilocarpic acid diester (1.5 pmol) or free base of bispilocarpic acid diester (3.0 pmol) were dissolved into 25.0 and 50.0 mL of tris buffer (0.05 M, pH 7.401, respectively. Hydrolysis of the bispilocarpic acid diesters was studied by incubating 2000 pL of diester solution and 500 pL of corneal supernatant at 37 "C. At preselected times up t o 300 min, aliquots of 150 pL were withdrawn and mixed with 300 pL of ethanol to precipitate the soluble proteins and thereby terminate the reaction. The mixture was centrifuged, and 20 pL of clear supernatant was injected into the LC and analyzed for remaining diester by LC. The rate constants for degradation were calculated as they were in the case of the plasma studies, and the r2 values for the plots were 0.98-1.00. The protein concentration in the incubation solution varied between 0.44 and 0.51 mg/mL as determined by a dye-binding assay with bovine serum albumin a s the standard.33 I n Vitro Enzymatic Rate of Hydrolysis in Butyrylcholinesterase Solution-First, 2.5 pmol of the bispilocarpic acid diester was dissolved in 25.0 mL of preheated (37 "C) tris buffer (0.05M, pH 7.40). Butyrylcholinesterase (BuChE; 15 IU)was dissolved in 5.0 mL of preheated (37 "C) tris buffer (0.05 M, pH 7.40). A 5.0-mL sample of the diester (0.5 pmol) solution and a 5.0-mL sample of the enzyme solution (15 IU) were mixed, yielding a solution of 30 IU enzyme/ pmol substrate. The solution was kept in a waterbath a t 37 "C,and at preselected times up to 300 min, 200 pL of the contents of the vial were withdrawn and mixed with 400 pL of ethanol to precipitate the proteins and terminate the reaction. The mixture was centrifuged, and 20 pL of clear supernatant was injected into the LC and was analyzed for remaining diester by the same LC method. The rate constants for degradation were calculated as before, and the r2 values for the plots were 0.98-1.00. M e a s u r e m e n t of A p p a r e n t Partition Coefficient-The lipophilicities of the bispilocarpic acid diesters were obtained from the distribution of the compounds between 1-octanol and phosphate buffer.34 The apparent partition coefficients were determined a t pH 5.0 to avoid solubility problems a t pH 7.4. The phosphate buffer (0.16 M, pH 5.0, p = 0.5 adjusted with NaC1) and 1-octanol phases were saturated before use by stirring vigorously for 24 h a t room temperature. The determinations of prodrugs concentrations and the calculations of apparent partition coefficients were carried out as described previously.17
Results and Discussion Identification of Bispilocarpic Acid Diesters-The most prominent fragment ions with respect to relative intensity of the bispilocarpic acid diesters V-XI1 are listed in Table 1. All the mass spectra show a molecular ion [M+] with low intensity and display corresponding fragment ions like the bispilocarpic acid diesters described earlier.31 The elemental Journal of Pharmaceutical Sciences / 657 Vol. 84, No. 5, May 1995
Table 1-Relative Abundances of the Ions in the El Mass Spectra of the Bispilocarpic Acid Diesters V - XI1 Compound
[M+.]
Other Fragment Ions
V VI VII Vlll
590 (5%) 614 (1%) 642(4%) 604 (6%)
IX
628 (7%)
X XI XI1
656 (6%) 656 (3%) 670 (2%)
503 (8%),397 (19%),396 (85%),382 (20%),381 (95%),309 (llyo),209 (18%), 195 (45%),96 (78%),95 (100%) 408 (11O/0),393 (lo%),320 (29%),291 (l6%),209 (l8Y0),208 (llYo),207 (78%),163 (29%),121 (47%),113 (52%),96 (38%),95 (100%). 436 (lo%), 423 (22%),421 (loo%), 421 (13%),408 (18%), 407 (85%),335 (lo%),221 (52%),209 (12%)' 121 (13%),96 (86%),95 (50%). 517 (7%),411 (13%),410 (57%),396 (16%),395 (71%),339 (36%),323 YO), 209 (IS%),208 (6%),195 (48%),121 (20%),96 (56%), 95 (100%). > - - -, 423(19%),422 (76%),408 (16%),407 (73%),352 (lo%), 351 (40%),334 (lo%),209 (19%),208 (1 l%),207 (73%),163 (21%),121 (32%), 96 (55%).95 (100%). 543 (9%),437 (23%),436 (loo%),422 (22%),421 (86%),366 (lo%),365 (46%),221 (58%),209 (14%),121 (15%),96 (68%),95 (78%) 450 (39%),435 (23%),379 (13%),363 (13%),209 (15%),207 (29%),163 (ll%),121 (23%),96 (50%),95 (100%). 464 (13),449 (lo%), 377 (lo%),209 (ll%),207 (21%),163 (l6%),121 (26%),96 (53%),95 (100%).
Table 2-Measured and Calculated Accurate Mass of Bispilocarpic Acid Diesters ~~~
Compound
Observed Mass
Calculated Mass
Error (mmu)
V VI VII Vlll IX X XI XI1
590.3363040 614.3299260 642.3648680 604.3491210 628.3428340 656.3743290 656.3775330 670.3941500
590.3315650 614.3315650 642.3628652 604.3472151 628.3472151 656.3785152 656.3785152 670.3941653
4.7 1.6 2.0 1.9 4.4 4.2 1.o 0.0
~
Elemental composition (free base)
Table 3-Proton Chemical Shifts for Compounds V-XI1 Chemical Shift (ppm) Proton
V
VI
VII
Vlll
IX
X
Xi
XI1
N-Me 2 4 6a 6b 7 8 9 10 11
3.61 7.51 6.73 2.67 2.67 2.31 2.50 1.67 0.90 4.08 4.34
3.63 7.58 6.78 2.69 2.69 2.33 2.50 1.69 0.92 4.08 4.35 1.61 0.90 0.90 -
3.79 8.50 7.23 2.77 2.77 2.40 2.54 1.71 0.93 4.11 4.36 3.16 2.22 2.01 1.89 6.73
3.62 7.56 6.76 2.69 2.69 2.33 2.49 1.68 0.91 4.08 4.18 2.01 2.31 1.10 1.10 -
3.61 7.51 6.73 2.68 2.68 2.33 2.49 1.68 0.91 4.07 4.19 2.02 1.62 0.90 0.90 -
3.80 8.50 7.23 2.78 2.78 2.40 2.53 1.71 0.92 4.11 4.21 2.03
3.77 8.34 7.15 2.77 2.77 2.38 2.51 1.69 0.91 4.09 4.14 1.72 1.48 1.61 0.88 0.88
3.81 8.53 7.24 2.79 2.79 2.38 2.52 1.69 0.91 4.09 4.12 1.69 1.45 1.61 0.90 0.90 6.72
Riy
R2a R$ R$' R2Y Fum. a
-a
2.32 1.10 1.10 -
-
-
-
3.16 2.22 2.01 1.89 6.73
-
6.71
-, Not applicable.
composition of the molecular ion was determined by measuring the accurate mass of the molecular ion. The error between observed and calculated mass <4.7 mmu for diesters V-XI1 (Table 21, which reliably verifies the elemental composition of the bispilocarpic acid diesters. The NMR investigation, together with mass spectrometry, ensures correct identification of the new drug substances. The IH NMR data of the bispilocarpic acid diesters V-XI1 were consistent with their structure. IH assignments were based on the 'H-'H-correlated spectra (COSY) and are listed in Table 3. Enzymatic Hydrolysis of Bispilocarpic Acid D i e s t e r s in Different Vehicles-Bispilocarpic acid diesters are very stable towards chemical hydrolysis in the absence of enz y m e ~ . ' However, ~ in the presence of esterases, prodrugs will be hydrolyzed to the corresponding monoesters with a char658 /Journal of Pharmaceutical Sciences Vol. 84, No. 5, May 1995
Rate Constant ( h - h g protein)
Log P a t
~
Rla RIB
Table 4-Apparent Partition Coefficient at pH 5.0 and Rates of Hydrolysis of Various Bispilocarpic Acid Diesters in 80% Human Plasma (pH 7.40), Homogenates of the Cornea of the Albino Rabbit (pH 7.40), and Butyrylcholinesterase Enzyme Solution (pH 7.40) at 37 "C
Prodrug
pH 5.0 (n = 3)
I II 111 IV V VI VII Vlll IX X XI XI1
-0.29 i 0.01 0.66 ? 0.05 1.69f 0.01 1.03i0.07 -0.77 i 0.04 -0.58 i0.01 0.30 i 0.01 -0.61 i 0.01 -0.30 i0.03 0.60 i 0.01 0.40 f 0.01 0.88 f 0.02
80% Human Plasma (n = 3)
Rabbit Cornea Butylylcholinesterase Homogenate Enzyme Solution (n = 2-4) (n = 3)
0.0076 f 0.00115 2.2 ?k 0.28 0.0086 i 0.00018 10.5 f 0.34 0.0055i0.00067 12.6 i2.05 0.0037 i- 0.00006 2.2 i 0.13 0.0166 L 0.00056 2.7 i 0.39 1.3 i 0.03 0.0016 f 0.00027 0.0081 f 0.00044 9.3 i 1.62 3.6 i 0.42 0.0093 f 0.00008 0.0008 i 0.00001 2.3 i 0.35 0.0094 f 0.00061 10.6i 1.13 0.0037 L 0.00007 1.7 i 0.19 0.0036 i 0.00031 2.5 i 0.03
1.3 2 0.05 8.1 f 0.04 15.1 f 2.24 1.8 f 0.06 5.1 i 0.68 0.8 f 0.01 15.4 f 0.39 4.5 f 0.17 0.5 f 0.02 14.5 f 2.52 0.9 i 0.05 0.7 i 0.02
NOTE: Results are expressed as mean f SE.
aderistic rate. The rate constants (normalized to the amount of proteins in t h e incubation mixture) for the hydrolysis of bispilocarpic acid diesters in diluted human plasma, rabbit cornea homogenate, and BuChE solution are listed in Table 4. The true comparison of rate constant of the same compound in different vehicles is not meaningful because the total activity of esterases and the maximum velocity (Vmm)and Michaelis constant (K,) values of diesters may vary in these systems. The substrate concentration in diluted human plasma varied from 100 to 250 pM (pH 7.4) and from 50 to 500 pM (pH 6.0) depending on the solubility and UV absorption of the prodrugs. At low substrate concentrations ([S] < 0.1 Km),the absolute rate of enzymatic hydrolysis is linearly dependent on the substrate concentration and the reaction follows firstorder kinetics. In this case, half-time is independent of the substrate concentration. The K , value was calculated to be 1.6 mM (pH 7.4,37 "C) for derivative I1from the LineweaverBurk plot (Figure 3), and the half-life (~-112) was independent of the substrate concentration in plasma hydrolysis. The K, was not determined for hydrolysis in cornea homogenate and i n BuChE solution. To confirm t h a t substrate concentration does not affect the t l l z of the diester, the same substrate concentration was used in every hydrolysis determinations in cornea homogenate and BuChE solution for each prodrug. The effect of substrate concentration on the rate of hydrolysis i n BuChE solution is shown in Table 5 . This result confirms that if the relationship [prodrugl/[enzymel is not small enough, the substrate concentration affects the value of tllz and thus the comparisons of the tllz values obtained from different laboratories are not reasonable.
y = 0 21003 + 3 4 1 . 4 1 ~ R ^ 2
=
Table 6-Half-Lives of Selected Bispilocarpic Acid Diesters in 80% Human Plasma Solution (pH 6.0) at 37 "C
0 990
f112 (min)
t112
Compd
Plasma (Batch A)
Plasma (Batch B)a
I I1 111 IV
13 5 4 26
46 42 19 269
a
Compd Xlll XIV
xv
(min)
Plasma (Batch A)
Plasma (Batch B)d
34 1 7 3
735 46 10
Plasma batch B is similar to plasma used in experiments at pH 7.4 in Table
4.
0 005
0 000
0010
0005
0 L15
0020
I/ISI tpM-')
Figure 3-Lineweaver-Burk plot of l/vo versus l/[S] for prodrug It initial velocity of enzymatic hydrolysis at diester concentration [q).
(yo
= the
Table 5-Effect of Relation BuChE Activity to the Half-Time of Derivative II BuChE Activity (luipmol substrate) 3
9 15
hi2
(min)
Degradation was not observed 52 23
BuChE Activity (IUlpmol substrate)
tIi2(min)
15
23
27 59
10 3.5
The derivatives 11, 111, V, VII, VIII, and X are good substrates for BuChE and they are also hydrolyzed rapidly in human plasma and rabbit cornea homogenate (Table 4). Consequently, these prodrugs will be hydrolyzed primarily by BuChE also in the cornea and plasma. The rate of hydrolysis by BuChE increased with increasing chain length of acyl substituent a t Rz (I, 11, 111). BuChE is the dominant esterase in rabbit ocular tissues.27 For example PGFzu esters are hydrolyzed by BuChE but not by acetylcho. ~ ~ naphthyl esters with linesterase or carbonic a n h y d r a ~ e The ester chains longer than four carbons are primarily hydrolyzed by BuChE, and for 1- and 2-naphthyl esters, the maximal hydrolytic rates by BuChE were reached at valerate (C5) and at butyrate (C4) esters, r e s p e c t i ~ e l y .A ~ ~similar parabolic chain length dependence of hydrolysis of 1- and 2-naphthyl esters in rabbit ocular tissue has also been reported.36 Thus, the ocular prodrug should preferably have a butyryl portion as an acyl group t o hydrolyze rapidly by BuChE in the eye. On the other hand, optimal prodrug ester should be hydrolyzed by several esterases to minimize the effect of individual fluctuations in the proportion of esterases in the eye.37 The results of the hydrolysis studies show that the rate of enzymatic hydrolysis is greatly dependent on the ester structure in every vehicle. The most stable derivatives in this series are the compounds that have the cyclopropyl as Rz (IV, VI,M,XI, and XII). This result is in good agreement with other e n z y m a t i ~and ~ ~c~h~e ~m i ~ a lhydrolysis ~ ~ , ~ ~ studies of prodrugs. The stability of the cyclopropanoyl ester is due to steric effects of the cyclic side chain.39 It is evident that increased branching of the alkyl group in the acyl portion of the prodrug esters (e.g., pivaloyl and isopropyl esters) decrease
the rate of enzymatic The most labile derivatives in this series for enzymatic hydrolysis are 0,O'dicyclobutane derivatives (Table 4; VII, X), which may be due t o the flexibility of the cyclobutane structure that may decrease the steric hindrance to attack by esterases. Although the RZ moiety of bispilocarpic acid diesters controls the rate of enzymatic conversion to the corresponding bispilocarpic acid monoesters, and the structure of the R1 determines the rate of chemical hydrolysis t o p i l o ~ a r p i n e , ~ ~ ~ ~ variations in the R1 moiety may also have an effect on the rate of enzymatic hydrolysis (Table 4). For example, prodrugs IV,VI, IX,XI, and XI1 have the same Rz, but R1 is varied; this change in R1 causes variations in the rate of enzymatic hydrolysis, especially in BuChE solution and in diluted human plasma. Thus, both pro-moieties of double prodrugs may have a n effect on the reaction that affects only the other pro-group. This conclusion is not in agreement with the studies on corresponding pilocarpic acid diesters.l6Yz1 Possibly the R1 group has a more remarkable effect on the conformation of bis-structure than on mono-structure. On the other hand, in prodrugs V, VIII, VII, and X, R2 is similar but Rl is varied and the rates of enzymatic hydrolysis were similar. Thus, the effect of R1 structure on the rate of enzymatic hydrolysis of R2 is also dependent on the structure of Rz. Effect of pH on Enzymatic Hydrolysis in Human Plasma-The bispilocarpic acid diesters are very lipophilic weak bases (pK, x 7)17J8that are poorly water soluble at pH 7.4. Because of this poor water solubility, hydrolysis studies a t pH 7.4 with HPLC determination suffer from sensitivity problems. We studied enzymatic hydrolysis at pH 6.0 in diluted human plasma to decrease the solubility problems. The rate of enzymatic hydrolysis a t pH 6.0 was clearly slower than the corresponding value at pH 7.4; therefore, it is not reasonable to use pH 6.0 instead of 7.4. This slow rate of hydrolysis may be due to the protonation of prodrugs, which leads to structural and electrostatic changes.43 This slow rate of hydrolysis at pH 6.0 may also suggests that whereas the uncharged form of the bispilocarpic acid diesters is hydrolyzed enzymatically the protonated form is not. The slight decrease of cholinesterase activity at pH 6.044may have only a minor effect on the rate of enzymatic hydrolysis. During the enzymatic hydrolysis studies at pH 6.0, we found that the results can vary widely depending on plasma batch even though we used pooled plasma. The half-lives of enzymatic hydrolysis of some selected derivatives in two different plasma batches are presented in Table 6. Major variations in plasma may be due to different patient groups. Although the final explanation was not found, this result indicates that the whole analogy series must be studied in the same plasma or serum batch and the results from different laboratories can be dependent on the enzyme systems used. Related problems are involved when eye homogenate is used because the esterase activity in rabbit eye changes with the tissue,45 age,46s47and p i g m e n t a t i ~ n . ~ ~ Effect of Lipophilicity of the Prodrug on Enzymatic Hydrolysis-All bispilocarpic acid diesters are more lipophilic Journal of Pharmaceutical Sciences / 659 Vol. 84, No. 5, May 1995
than the parent compound, pilocarpine (log P = -1.72 at pH 5.01,mainly because of replacement of the lactone ring by ester groups (Table 4). Opening and esterification of the lactone ring have no effect on the ionization of the imidazole moiety of bispilocarpic a ~ i d , ~and, ~ . ' thus, ~ the observed changes in lipophilicity are not due to the decrease in t h e pK,. It has also been shown that the drug-protein binding generally increases with increasing partition coefficient of a For example, protein binding of pilocarpic acid monoesters increases with increasing partition coefficient.21 Thus, it is possible that the rates of enzymatic hydrolysis of lipophilic prodrugs become slower because of strong protein binding especially if the in uitro experiments have been done in vehicles with high protein concentration (plasma, serum). The effect of protein binding on enzymatic hydrolysis is difficult to clarify by determining the rate of hydrolysis K,) of substrates in because all other properties (e.g., V, whole series should be constant. However, the results in Table 4 show that increased lipophilicity of bispilocarpic acid diesters and possibly related protein binding do not have clear effects on the enzymatic hydrolysis.
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Acknowledgments This study was supported by a grant from Leiras Oy (Finland) and the Technology Development Centre (Finland). Art0 Urtti was supported by the Academy of Finland. Ms. Paivi Varis and Mrs. Marja Mali is gratefully acknowledged for her skillful technical assistance.
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