Kinetics of the esterification of active pharmaceutical ingredients containing carboxylic acid functionality in polyethylene glycol: formulation implications.

Polyethylene glycols (PEGs) are attractive as excipients in the manufacture of drug products because they are water soluble and poorly immunogenic. They are used in various pharmaceutical preparations. However, because of their terminal hydroxyl groups, PEGs can participate in esterification reactions. In this study, kinetics of two active pharmaceutical ingredients, cetirizine and indomethacin possessing carboxylic acid functionality, has been studied in PEG 400 and PEG 1000 at 50 °C, 60 °C, 70 °C, and 80 °C. HPLC-UV was applied for the determination of concentrations in the kinetic studies, whereas HPLC-MS was used to identify reaction products. The esterification reactions were observed to be reversible. A second-order reversible kinetic model was applied and rate constants were determined. The rate constants demonstrated that cetirizine was esterified about 240 times faster than indomethacin at 80 °C. The shelf-life for cetirizine in a PEG 400 formulation at 25 °C expressed as t(95%) was predicted to be only 30 h. Further, rate constants for esterification of cetirizine in PEG 1000 in relation to PEG 400 decreased by a factor of 10, probably related to increased viscosity. However, it is important to be aware of this drug-excipient interaction, as it can reduce the shelf-life of a low-average molecular weight PEG formulation considerably.

Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non-tumour brain tissue.

The endogenous levels of the two cannabinoid receptor ligands 2-arachidonoyl glycerol and anandamide, and their respective congeners, monoacyl glycerols and N-acylethanolamines, as well as the phospholipid precursors of N-acylethanolamines, were measured by gas chromatography-mass spectrometry in glioblastoma (WHO grade IV) tissue and meningioma (WHO grade I) tissue and compared with human non-tumour brain tissue. Furthermore, the metabolic turnover of N-acylethanolamines was compared by measurements of the enzymatic activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolysing phospholipase D and fatty acid amide hydrolase in the same three types of tissue. Glioblastomas were characterized by enhanced levels of N-acylethanolamines (eightfold, 128 +/- 59 pmol/micromol lipid phosphorus) including anandamide (17-fold, 4.6 +/- 3.1 pmol/micromol lipid phosphorus) and several species of N-acylphosphatidylethanolamines (three to eightfold). This was accompanied by a more than 60% reduction in the enzyme activities of N-acylphosphatidylethanolamine-hydrolysing phospholipase D and fatty acid amide hydrolase. By contrast, meningiomas were characterized by a massively enhanced level of 2-monoacyl glycerols (20-fold, 2293 +/- 361 pmol/micromol lipid phosphorus) including 2-arachidonoyl glycerol (20-fold, 1524 +/- 361 pmol/micromol lipid phosphorus). This was accompanied by an enhanced in vitro conversion of phosphatidylcholine to monoacyl glycerol (fivefold). The enhanced level of the 2-arachidonoyl glycerol, anandamide and other N-acylethanolamines detected in the two types of tumour tissue may possibly act as endogenous anti-tumour mediators by stimulation of both cannabinoid and non-cannabinoid receptor-mediated mechanisms.

Brain levels of N-acylethanolamine phospholipids in mice during pentylenetetrazol-induced seizure.

The N-acylethanolamine phospholipids (NAPE) are precursors for N-acylethanolamines (NAE), including anandamide (20:4-NAE), which is a ligand for the cannabinoid receptors. Previously, NAPE were believed to be found only in injured tissue, e.g., after neurodegenerative insults. Neuronal injury may occur in response to seizure activity. Therefore, we investigated the effect of pentylenetetrazol (PTZ)-induced seizures in PTZ-kindled mice on the level of NAPE in the brain. Male NMRI mice were kindled with PTZ injections 3 times/wk, thereby developing clonic seizures in response to PTZ. Mice were killed within 30 min after the clonic seizure on the test day (12th injection) and the brains were collected. Eight species of NAPE were analyzed as the glycerophospho-N-acylethanolamines by high-performance liquid chromatography-coupled electrospray ionization mass spectrometry. No effect of the PTZ kindling on the NAPE levels in murine brains was observed. Total NAPE in control mice cortex (n = 4) was 16.4 +/- 3.0 micromol/g wet weight of which 20:4-NAPE accounted for 3.6 mol%, and the major species was 16:0-NAPE, accounting for 52.1 mol%. Determination of the activity of NAPE-hydrolyzing phospholipase D and of N-acyltransferase in brain membrane preparations from adult and 3-d-old mice revealed an enzyme pattern in the adult mice that was favorable for NAE accumulation as opposed to NAPE accumulation. Thus, there was no difference in NAPE levels; at present, however, this does not exclude that NAE may accumulate during seizure.

Putative neuroprotective actions of N-acyl-ethanolamines.

N-Acyl-ethanolamines (NAEs) and their precursors, N-acyl-ethanolamine phospholipids (NAPEs), are present in the mammalian brain at levels of a few hundred picomoles/gram tissue and a few nanomoles/gram tissue, respectively. NAE-containing arachidonic acid is called anandamide, and it has attracted particular attention since it is a partial agonist for the cannabinoid receptors, for which 2-arachidonoylglycerol is the full agonist. In addition, anandamide may also activate the vanilloid receptor. Anandamide usually amounts to 1-10% of NAEs, as the vast majority of N-acyl groups are saturated and monounsaturated fatty acids. Formation of NAPE and NAE is catalyzed by an N-acyltransferase and an NAPE-hydrolyzing phospholipase D, respectively, two enzymes that have been characterized only preliminary. Interestingly, NAPEs and NAEs accumulate in the brain in response to neurodegenerative insults at a time when other phospholipids are subjected to rapid degradation. This is an important biosynthetic aspect of NAPE and NAE, as NAEs may be neuroprotective by a number of different mechanisms involving both receptor activation and non-receptor-mediated effects, e.g. by binding to cannabinoid receptors and interfering with ceramide turnover, respectively.

Substantial species differences in relation to formation and degradation of N-acyl-ethanolamine phospholipids in heart tissue: an enzyme activity study.

The formation of N-acyl-ethanolamines (NAEs), including the cannabinoid receptor ligand anandamide, and their precursors N-acyl-ethanolamine phospholipids (NAPEs) are catalyzed by NAPE-hydrolyzing phospholipase D (NAPE-PLD) and N-acyl-transferase, respectively. NAPE and NAE are suggested to have beneficial effects on the heart, but in the literature there are indications of species differences in the activity of these enzymes. We have examined heart microsomes from rats, mice, guinea pigs, rabbits, frogs, cows, dogs, cats, mini pigs and human beings for activities of these two enzymes. N-Acyl-transferase activity was very high in dogs and cats (>13 pmol/min/mg protein) whereas it was very low to barely detectable in the other species (<3 pmol/min/mg protein). NAPE-PLD activity was very high in rats and guinea pigs (>45 pmol/min/mg protein) whereas it was 9 pmol/min/mg protein in frogs and below that in the other species. The ratio of activity between the two enzymes varied from 0.002 to 15 in the investigated species. The activity of the two enzymes in rat hearts as opposed to rat brain did not change during development. These results indicate that there may be substantial species differences in the generation of anandamide and other NAEs as well as NAPEs in heart tissues.