Pilocarpine Prodrugs Research Articles

Modified β-Cyclodextrin (SBE7-β-CyD) with Viscous Vehicle Improves the Ocular Delivery and Tolerability of Pilocarpine Prodrug in Rabbits

The complexation of pilocarpine prodrug, O,O'-dipropionyl-(1,4-xylylene) bispilocarpate, with various beta-cyclodextrin (beta-CyD) derivatives was studied by the phase solubility method. The effects of coadministered sulphobutyl ether beta-CyD (SBE7-beta-CyD) with and without poly(vinyl alcohol) (PVA) on the miotic response and eye irritation of the prodrug were investigated in pigmented rabbits. The pilocarpine prodrug formed 1:1 inclusion complexes with variably substituted sulphobutyl ether derivatives of beta-CyD (SBE4-beta-CyD and SBE7-beta-CyD), and 1:1 and 1:2 complexes with hydroxypropyl-beta-CyD (HP-beta-CyD) at pH 7-4. Coadministered SBE7-beta-CyD eliminated the eye irritation due to the pilocarpine prodrug, but also decreased the miotic response. Ocular absorption of the prodrug was improved by increasing the viscosity of prodrug/SBE7-beta-CyD solution with PVA without inducing any eye irritation. Eye irritation due to viscous prodrug/SBE7-beta-CyD solutions was comparable with isotonic NaCl solution. We conclude that administration of pilocarpine prodrug in viscous SBE7-beta-CyD solution decreases substantially eye irritation while ocular absorption is not affected.

Miotic effect and irritation potential of pilocarpine prodrug incorporated into a submicron emulsion vehicle

Pilocarpine prodrug, O, O′-dipivaloyl(1,2-ethylene) bispilocarpic acid diester, was introduced to a submicron emulsion vehicle in a dose equivalent to 0.5% pilocarpine base, and the formulation was studied in albino rabbits using miotic assay. Compared with pilocarpine HCl 0.5% solution delayed and prolonged miosis was observed after application of the prodrug emulsion. AUC 0–6 h values for the prodrug emulsion and pilocarpine solution were 9252±1345 and 6845±1967%×min, respectively. The prodrug was also administered twice daily for 5 days in the form of aqueous solution or submicron emulsion in order to study ocular irritation. Irritation potential of the prodrug was significantly reduced when submicron emulsion was used as a vehicle.

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

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,O′-(1,4-xylylene) bispilocarpic acid diester prodrugs [O,O′-diacetyl (III), O,O′-dipropionyl (IV) and O,O′-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 2.8×10 −6 cm/s to 0.9×10 −6 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.

β-Cyclodextrin derivatives (2-HP-β-CD, SBE4-β-CD) decrease the amphiphilicity and membrane perturbing effects of pilocarpine prodrugs

The ocular bioavailability of pilocarpine has been improved by bispilocarpic acid diesters but the ocular irritation associated with these prodrugs is the main limitation of their clinical usefulness. Pilocarpine prodrugs show amphiphilic lipid bilayer disrupting properties which partly contribute to the eye irritation. In the present study, the influence of complexation with hydrophilic β-cyclodextrin derivatives, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether β-cyclodextrin with an average degree of substitution of four (SBE4-β-CD), on pilocarpine prodrug-induced bilayer perturbation was investigated using rabbit erythrocyte hemolysis and calcein release from liposomes as test models. Bispilocarpic acid diesters form 1:1 and 1:2 complexes with HP-β-CD and 1:1 complexes with SBE4-β-CD at pH 7.4. Both β-CD derivatives decreased the surface activity of the prodrug solutions. The hemolytic activity and liposome disruption of prodrug-CD solutions were 3–10 times and 4–15 times lower than that of parent prodrug, respectively. The protecting effect of CDs was dependent on the apparent complexation constant of prodrug/CD complex which determines the active free prodrug concentration in CD solution. The present study shows that amphiphilic properties and lipid bilayer perturbing effects of pilocarpine prodrugs are reduced by complexing them with hydrophilic β-CD derivatives.

β-CD derivatives decrease the membrane perturbing effects of pilocarpine prodrugs

β-CD derivatives decrease the membrane perturbing effects of pilocarpine prodrugs

Amphiphilic properties of pilocarpine prodrugs

Corneal permeability of pilocarpine has been increased by prodrug derivatization, but ocular irritation is associated with the in vivo use of the prodrugs. To determine possible amphiphilic nature of the prodrugs, we determined the critical micelle concentrations and lipid bilayer disrupting properties of bispilocarpic acid diester prodrugs. Surface activity of prodrugs in water was determined using interfacial tensiometer. Lipid bilayer disruption was tested by calcein release from liposomes and hemolysis of rabbit erythrocytes in vitro. Molar phospholipid and cholesterol contents of the liposomes (EPC:DOPE:DPPG:Ch 8:6:1.5:1.5) were designed on the basis of the lipid content of the corneal epithelium. Surface activities of the pilocarpine prodrugs increased with the increasing lipophilicity of the derivative and increasing pH. The critical micelle concentrations of the prodrugs were 0.1-1 mM at pH 5.0. The concentrations of the prodrugs to induce 50% leakage of calcein were 0.5 mM (V), 1.2 mM (III), 2.2 mM (IV) and 0.25 mM (V), 0.8 mM (III), 3.5 mM (IV) to cause 50% hemolysis. Also, eye irritation of these prodrugs increased with their increasing lipophilicity, pH and concentration. Lipophilic pilocarpine prodrugs show amphiphilic properties which may contribute to the eye irritation. The harmful effects of pilocarpine prodrugs on cell membranes on ocular surface may limit their usefulness. Possible amphiphilicity of lipophilic prodrugs may be a limitation of the prodrug technique in ocular drug delivery.

Comparison of Enzymatic Hydrolysis of Pilocarpine Prodrugs in Human Plasma, Rabbit Cornea, and Butyrylcholinesterase Solutions

Various bispilocarpic acid diesters (double prodrugs of pilocarpine) were synthesized, and theirin vitroesterase 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.

Sulfobutyl Ether β-Cyclodextrin (SBE-β-CD) in Eyedrops Improves the Tolerability of a Topically Applied Pilocarpine Prodrug in Rabbits

The effects of a novel, modified beta-cyclodextrin (SBE4-beta-CD; a variably substituted sulfobutyl ether with an average degree of substitution of four) on eye irritation and miotic response of an ophthalmically applied pilocarpine prodrug, O,O'-dipropionyl-(1,4-xylylene) bispilocarpate, in albino rabbits were studied. Compared to the commercial pilocarpine eyedrop solution (163 mM, equivalent to 3.4% pilocarpine), 12-24 mM pilocarpine prodrug solutions (equivalent to 0.5-1.0% pilocarpine, respectively) decreased peak miotic intensity (Imax) and increased the time to reach peak (tmax), but did not significantly affect values for the area under the miosis versus time curves (AUC), i.e. 12-24 mM pilocarpine prodrug appeared to be equivalent to 163 mM pilocarpine. Ocularly applied 12-24 mM pilocarpine prodrug solutions, however, were more irritating than a commercial pilocarpine eyedrop solution. Coadministered SBE4-beta-CD significantly decreased the eye irritation of the pilocarpine prodrug solutions. Coadministered SBE4-beta-CD did not affect the miotic response of prodrug solution when the molar ratio of SBE4-beta-CD to prodrug was low. However, increasing the molar ratio of SBE4-beta-CD to prodrug decreased the Imax and AUC values. The results show that eye irritation of the pilocarpine prodrug is prevented by levels of SBE4-beta-CD that do not affect the apparent ocular absorption of the prodrug.

Ocular absorption and irritation of pilocarpine prodrug is modified with buffer, polymer, and cyclodextrin in the eyedrop.

The influence of buffer, viscosity and cyclodextrin on the ocular absorption and irritation of a pilocarpine prodrug, O,O'-dipropionyl-(1,4-xylylene) bispilocarpic acid diester, was studied in albino rabbits. The prodrug solutions, equivalent to 0.5% pilocarpine, were prepared in 0, 10, 20, 50, or 75 mM citrate buffer at pH 5.0. Viscosity of the solutions (20, 50 or 115 cP) was modified with hydroxypropyl methylcellulose. 2-hydroxypropyl-beta-cyclodextrin (HPCD) was included at concentrations 5, 10 and 15% (w/v). The formulations were compared to a commercial pilocarpine eyedrop (1.7%). Ocular irritation was graded in a double-masked experiment and miosis was used as a bioassay for pilocarpine delivery to the iris. The prodrug showed decreased peak and prolonged duration of miosis compared to pilocarpine, but it caused ocular irritation. Increasing buffer strength decreased and elevated viscosity intensified the miotic response and irritation by the pilocarpine prodrug. HPCD decreased both the ocular delivery of pilocarpine and the irritation by the prodrug, but the net effect was positive. Thus, administering 1.0% of pilocarpine as a prodrug with 15% (w/v) HPCD, the irritation was at the same level with the commercial pilocarpine eyedrop, but the ocular delivery was substantially improved. In conclusion, the ocular delivery of the pilocarpine prodrug may be enhanced in relation to its local irritation by properly combining buffer, viscosity and HPCD.

Hydroxypropyl-beta-cyclodextrin increases the aqueous solubility and stability of pilocarpine prodrugs.

The effects of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. The solubility of prodrugs was studied by phase-solubility method (0-72.5 mM HP-beta-CD). The stability of one of the prodrugs was investigated as a function of temperature (40 degrees C-70 degrees C) and HP-beta-CD concentration (0-72.5 mM). The apparent rate constants (k1, k2) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K1:1, K1:2) were calculated by the curve-fitting method. The phase-solubility diagrams were classified as Ap-type and the apparent stability constants (K1:1, K1:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143-815 M-1 and 29-825 M-1, respectively. The stability of prodrug increased as a function of HP-beta-CD concentration over the studied temperature range. The shelf-life (t90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP-beta-CD solution increased 5.1-fold and 6.1-fold at 25 degrees C and 4 degrees C, respectively. The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the 1:2-complex than in the 1:1-complex and the relative amounts of complex species were found to be dependent on CD concentration.

A theoretical investigation of some novel pilocarpine prodrugs

AbstractA new class of pilocarpine double prodrugs of the bispilocarpic acid diester type has been investigated. Molecular orbital and molecular mechanics calculations have been done to obtain information about the conformational preferences and general physicochemical properties. Geometries of the structures were obtained with the force field implemented in the CHEM‐X program and using the semiempirical quantum chemical program AMPAC and the AM1 method. A correlation between the van der Waals volume, van der Waals area, and log P of the bispilocarpic acid diester structures is observed. The structures of these biscompounds are compared with the structures of the corresponding monopilocarpic acid diesters. Electrostatic and electronic properties of the compounds will be discussed in the context of the interaction between the prodrugs and the hydrolyzing esterase enzymes. © 1994 John Wiley & Sons, Inc.

Water soluble pilocarpine prodrugs with sustained intraocular activity in normotensive rabbits and in glaucomatous beagles

Water soluble pilocarpine prodrugs with sustained intraocular activity in normotensive rabbits and in glaucomatous beagles

Bispilocarpic acid monoesters as prodrugs of pilocarpine: II. Physicochemical properties and kinetics of hydrolysis in aqueous solution

The physicochemical properties and degradation kinetics of bispilocarpic acid monoesters were studied in order to assess the suitability of these compounds as prodrugs of pilocarpine. The esters show variable increases in lipophilicity relative to pilocarpine as evaluated by drug partitioning between 1-octanol and phosphate buffer at pH 7.40 and by the capacity factor ( k') of reversed-phase liquid chromatography. The degradation kinetics of the compounds were studied in aqueous solutions as a function of pH and temperature to determine their stability and suitability as prodrugs of pilocarpine. The bispilocarpic acid monoesters were hydrolyzed to yield pilocarpine in quantitative amounts in aqueous solution. Although bispilocarpic acid monoesters showed maximal stability in acidic solutions, the shelf-lives at pH 4.20 and at 4°C were less than 10 days. Consequently, it may be difficult to prepare ready-to-use aqueous solutions at acceptable pH (pH > 4.0).

Synthesis and analysis of O,O'-dicarboxylate (dibenzyl) bispilocarpates as possible prodrugs of pilocarpine.

As a part of a series of studies to develop prodrug derivatives of pilocarpine, the O,O'-succinyl (dibenzyl), O,O-adipoyl (dibenzyl), O,O-fumaryl (dibenzyl), and O,O-terephthaloyl (dibenzyl) bispilocarpate fumarates were synthesized as a new class of pilocarpine prodrugs. The compounds were prepared from pilocarpic acid benzyl monoester by coupling two pilocarpic acid benzyl monoesters together with spacer chains by usual esterification methods. Liquid chromatography, thermospray liquid chromatography-mass spectrometry, high-resolution mass spectrometry, and NMR spectroscopy were applied to the identification and the purity evaluation of the synthetic products.

Bispilocarpic acid monoesters as prodrugs of pilocarpine: I. Preparation and identification

Several alkyl and aralkyl bispilocarpic acid monoesters were synthesized as a new class of pilocarpine prodrugs in order tc minimize the formation of pro-moiety per mole of active pilocarpine. The compounds were prepared from pilocarpic acid by the usual esterification methods. The yield of the synthesis varied from 34 to 95%. Liquid chromatography (LC), thermospray-liquid chromatography-mass spectrometry (TSP-LC-MS), electron ionization-mass spectrometry (EI-MS) and NMR spectroscopy were applied for the identification and purity evaluation of the synthetic products.

OO′-(1,4-xylylene) bispilocarpic acid esters as new potential double prodrugs of pilocarpine for improved ocular delivery. I. Synthesis and analysis

An analogue series representing novel diesters of bispilocarpic acid, O, O′-( l,4-xylylene) bispilocarpic acid esters, were synthesized as double prodrugs of pilocarpine in order to improve the ocular delivery characteristics of the drug. In previous studies, various bispilocarpic acid monoesters have been synthesized and evaluated as prodrugs of pilocarpine. However, these derivatives suffer from instability in aqueous solution and hence were not suitable as a pilocarpine prodrug. Based on earlier results, 1,4-xylylene bispilocarpate was selected as the starting material for the synthesis of diesters. Bispilocarpic acid diesters were prepared by esterifying the free hydroxyl groups of bispilocarpic acid monoesters. All the diesters formed a salt with 3 equivalents of fumaric acid. The yields of bispilocarpic acid diester fumarates varied from 48 to 99%. The identification of the compounds and evaluation of the purity of synthetic products were performed by liquid chromatography with UV detection, thermospray liquid chromatography mass spectrometry, electron impact ionization mass spectrometry and 1H-NMR spectroscopy. The elemental composition of each compound was determined on high-resolution mass spectrometry by measuring the accurate mass of the molecular ion.

O, O′-(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

Various O, O′-(1,4-xylylene) bispilocarpic acid esters, i.e. bispilocarpic acid diesters, were evaluated as water-soluble double prodrugs of pilocarpine. All the prodrug derivatives (log P = 2.76–7.03) were more lipophilic than pilocarpine (log P = 0.01) asg determined from partitioning between 1-octanol and buffer (pH 7.40) or from LC capacity factors. The bispilocarpic acid diester fumarates were shown to be more water-soluble prodrugs than previously described pilocarpic acid diester fumarates. The aqueous stability of the derivatives was investigated as a function of pH and temperature. Maximal stability was achieved in acidic solutions. The shelf-life of O, O′-dipropionyl (1,4-xylylene) bispilocarpate fumarate was 469 days at pH 6.0 and 4 ° C. Hence, the bispilocarpic acid diester prodrugs possess sufficient aqueous stability to allow formulation of ready-to-use solutions. The diesters were hydrolyzed enzymatically to yield bispilocarpic acid monoester which cyclized to the parent pilocarpine in quantitative amounts. The half-lives of diesters in human plasma varied from 2 to 94 min, being highly dependent on the ester group. It appears that bispilocarpic acid diesters are a promising group of new pilocarpine prodrugs that offer possibilities from the results in stability, solubility, lipophilicity, and enzymatic hydrolysis tests.

Synthesis and identification of pilocarpic acid diesters, prodrugs of pilocarpine

A series of new pilocarpic acid diesters were synthesized to obtain prodrugs for pilocarpine with varying physico-chemical properties. Thermospray liquid chromatography-mass spectrometry (TSP-LC-MS), liquid chromatography with UV-detection (LC-UV) and NMR-spectroscopy were used for the identification of the synthetic products and for evaluation of their purity including typical impurities (pilocarpic acid monoester, pilocarpine). TSP-LC-MS-analysis was performed in the reversed-phase mode using acetonitrile (60%)-0.2 M ammonium acetate (40%) as mobile phase. In LC-UV-analysis chromatographic separation was carried out on a reversed-phase column and the mobile phase consisted of methanol (71%) and 0.02 M potassium dihydrogen phosphate, pH 4.5 (29%). Electron ionization-mass spectrometry (EI-MS) was used for elucidation of structures. Elemental compositions of the substances were verified with high resolution-mass spectrometry (HR-MS). The complete establishment of structures presented was based on 1H-, and COSY-NMR-spectroscopy joined to TSP-LC-MS-analysis.

Determination of physicochemical properties, stability in aqueous solutions and serum hydrolysis of pilocarpic acid diesters

New alkyl and aralkyl pilocarpic acid diesters, prodrugs of pilocarpine, were synthesized with the aim of improving the bioavailability of pilocarpine by increasing its corneal permeability. These esters were several orders of magnitude more lipophilic than pilocarpine as determined by their apparent partition coefficients between 1-octanol and phosphate buffer (pH 7.40) (log P). Good correlation between log P and HPLC capacity factors of the compounds was observed. All the compounds are stable in acidic aqueous solution; in serum, however, pilocarpic acid diesters are hydrolysed enzymatically to pilocarpic acid monoester, which undergoes spontaneous cyclization to active pilocarpine and inactive isopilocarpine. The half-lives of the diesters in serum varied from 6-232 min. In addition to the direct effects of the R2, R1 moiety had a remarkable effect on the rate of enzyme-catalysed hydrolysis taking place in moiety R2. The formed pilocarpine was analysed with a new HPLC method which allowed good resolution of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid. Rates for pilocarpine formation were both determined by experiment and calculated using the STELLA simulation programme with known degradation rate constants of pilocarpic acid diesters and monoesters. Since the simulations were in good agreement with the experimental results, it is concluded that STELLA simulation programme is useful in predicting pilocarpine formation.

Improved corneal pilocarpine permeability with O,O'-(1,4-xylylene) bispilocarpic acid ester double prodrugs.

O,O'-(1,4-Xylylene) bispilocarpic acid esters are pilocarpine prodrugs containing two pilocarpic acid monoesters linked with one pro-moiety. Each mole of prodrug forms two pilocarpine moles in the presence of esterases. Corneal uptake and permeability of various bispilocarpic acid diesters were investigated in vitro using isolated albino rabbit corneas. The permeability coefficient of pilocarpine was 2.8 x 10(-6) cm/sec, whereas for bispilocarpic acid diesters, despite their large molecular weights (between 638 and 722), permeability coefficients were 6.5-20.2 x 10(-6) cm/sec. Only pilocarpine, and no intact prodrug, was observed at the endothelial side. Corneal uptake was increased with increasing lipophilicity, but a parabolic relationship between the logarithm of the apparent partition coefficient (1-octanol-pH 7.4 phosphate buffer) (log PC) and the corneal permeability was noticed. Corneal permeability and the rate of enzymatic hydrolysis of the compounds correlated well. The corneal permeability of pilocarpine given as lipophilic bispilocarpic acid diester (log PC greater than or equal to 3) prodrugs seems to be controlled by the formation of pilocarpine in the corneal epithelium rather than by the absorption of prodrugs into the epithelium or their epithelium-stroma transport rate.