Pekka Suhonen

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-β-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 pro-drug, 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.

Increase in aqueous solubility, stability and in vitro corneal permeability of anandamide by hydroxypropyl-β-cyclodextrin

Abstract Arachidonylethanolamide (AEA), an endogenous ligand for the cannabinoid receptor, has a low aqueous solubility and an instability which hinder its use in aqueous formulations. In the present study, the effect of cyclodextrins (CDs) on the aqueous solubility, stability and in vitro corneal permeability of AEA was studied. The corneal penetration of AEA in HP-β-CD formulations was investigated in vitro by using isolated corneas of rabbits. The phase solubility diagram with HP-β-CD was classified as Ap-type and stability constants (K1:1 and K1:2) for 1:1 and 1:2 inclusion complexes were calculated to be 39 419 M −1 and 12 M −1, respectively. The phase solubility diagram of AEA with DIME-β-CD and HP-γ-CD were of the A l -type, indicating the formation of 1:1-complexes. The stability constants for 1:1-complexes were 744 877 M−1 and 15 469 M−1, respectively. The complexation of AEA with HP-β-CD markedly increased the stability of AEA. The shelf-life (t90%) of AEA in 10.0% HP-β-CD solution at 50°C was determined to be 166 days. The complexation of AEA with HP-β-CD increased corneal penetration of AEA compared to a suspension of the compound. Maximum permeability was achieved with the lowest HP-β-CD concentration that dissolved AEA completely. The permeability of AEA correlated well with the concentration of free AEA in solution.

Extracellular and intracellular factors influencing gene transfection mediated by 1,4-dihydropyridine amphiphiles

Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0-7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile-DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA-amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.

Permeability of pilocarpic acid diesters across albino rabbit cornea in vitro

Abstract Corneal uptake and permeability of various alkyl and aryl diesters of pilocarpic acid in isolated albino rabbit cornea were investigated in vitro in diffusion cells. The permeability coefficient for pilocarpine was 2.77 × 10 −6 cm/s. The fractional distribution of pilocarpine in the epithelial side, cornea and endothelial side at 4 h was 85.0, 11.0 and 4.0%, respectively. The corneal permeability coefficient of some pilocarpic acid diesters was several times higher (maximum 3.4-fold). No intact prodrug was observed in the endothelial side. 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 and the permeability of the most lipophilic derivatc was less than that of pilocarpine. In contrast, corneal uptake was increased with increasing lipophilicity being almost complete with a log PC value of 7.70. Corneal permeability and the rate of enzymatic hydrolysis of the compounds correlated well. The corneal permeability of lipophilic pilocarpine diesters ( log PC ≥ 2.87) given as prodrug 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. The optimal lipophilicity for improving corneal permeability (i.e. rate of ocular pilocarpine delivery), was observed at log PC values of 3–4, but more extensive corneal uptake by the most lipophilic compounds suggests that the largest ocular bioavailability may be obtained with larger values of log PC.

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

Abstract 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.

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

Abstract0,0′-(l,4-Xylylene) bispilocarpic acid esters are pilocarpine pro-drugs 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 × 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 × 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 ≥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.

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

Abstract 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.

Amphiphilic properties of pilocarpine prodrugs

Abstract 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.

Rate control of ocular pilocarpine delivery with bispilocarpic acid diesters

Abstract Ocular delivery of pilocarpine as bispilocarpic acid diester prodrugs was studied in albino rabbits using miosis as a bioassay of pilocarpine availability in the iris. Bispilocarpic acid diester fumarate eyedrops, in a dose equivalent to 0.5% pilocarpine, were administered at pH 5.0. Bispilocarpic acid diesters increased the duration of miosis and decreased the peak miotic response of pilocarpine. The time of peak miosis was delayed from 40 min to 55–135 min with the prodrugs. The duration of action was extended with seven compounds from 3 h to 4–5 h. Plateauing responses indicating sustained release of pilocarpine from the prodrug were seen in some cases. Compared with 1% pilocarpine, the prodrugs showed either increased, decreased or equal biphasic availability of pilocarpine in the iris. Neither lipophilicity nor enzymatic lability of prodrug alone could explain the miosis profile of pilocarpine. Eye irritation increased with increasing lipophilicity of the prodrugs. It appears that the ocular bioavailability of pilocarpine and its duration of action can be improved by bispilocarpic acid diesters, but in predicting their performance both lipophilicity and prodrug cleavage rate should be taken into account.

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.