Pekka Peura

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.

Concentrations of Tryptoline and Methtryptoline in Rat Brain

Abstract: Combined gas chromatography‐mass spectrometry and gas chromatography‐tandem mass spectrometry have been used to identify and quantify tryptoline, methtryptoline, 5‐hydroxytryptoline, and 5‐hydroxymethtryptoline as their heptafluorobutyryl derivatives in extracts of rat brain. Tryptoline and methtryptohne were identified on the basis of their retention times and mass spectral characteristics: they were reliably detected in brain tissue extracts without interference from artifactual formation; their whole brain concentrations ranged between 0.2 and 3 ng/g; and they had a similar neu‐roanatomical distribution, with the highest concentrations in the cerebellum and the cortex. Smaller quantities of 5‐hydroxytryptoline and 5‐hydroxymethtryptoline were also identified on the basis of their retention times and mass spectral characteristics. However, the significance of this finding is unclear, because these two compounds were accompanied by larger quantities of their tetradeuterated analogues formed from tetradeuterated‐5‐hydroxytryptamine added at the time of tissue homogenization; this result suggests that formation of 5‐hydroxytryptoline and 5‐hydroxymethtryptoline occurred during tissue homogenization, sample preparation, or both.

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.

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.

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.

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

Abstract 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.

Determination of tryptamine in rat brain by gas chromatography-mass spectrometry.

Tryptamine (TA) occurs in trace levels in the brain, but its role in the central nervous system is not clear. However, there is evidence that TA may be a neuromodulator since it binds to specific binding sites in the brain. TA was measured as a diheptafluorobutyryl derivative in rat whole brain by capillary gas chromatography-mass spectrometry using negative chemical ionization (NCI) and single ion monitoring (SIM). d(4)-TA was used as the internal standard. The ions m/z 532 and m/z 536 were monitored to identify TA and d(4)-TA, respectively and to calculate the concentration of TA in rat whole brain which was found to be 0.19 +/- 0.08 ng g(-1) (n = 8). The results confirm the earlier TA concentrations measured by GC-MS using positive electron impact ionization. However, NCI improved the signal/noise ratio of the method increasing its sensitivity for TA.